Integrity Initiatives in the Südhof Lab

Updated January 10, 2025

Südhof Lab Policies

As science has evolved over the last decades from a paper-based to a web-based endeavor, we have instituted procedures to enable transparent access to all data and tools of the Südhof lab beyond the documentation included in published papers. The overall lab policies are documented in our lab manual.

Current procedures include:

• Full unrestricted access to all published renewable reagents, including mice (via Jackson lab) and plasmids for non-profit research entities
• Access to all experimental protocols
• Public access to software applications (Apps) and algorithms (https://med.stanford.edu/sudhoflab/science-resources/tools.html)
• Public access to all raw data for published papers after 2023 in the publicly accessible Stanford Data Repository
• All manuscripts and published papers are screened for copy-paste errors and image artifacts using new A.I. tools
• Conversion of all lab notebooks to digital form (after 2023)
• Publication of all papers with an open access CC BY 4.0 license
• Publication of reviews of a paper with the paper when allowed by a journal
• Facilitating lab visits to disseminate key technologies

In addition, all experiments in the Südhof lab are required to conform to the following guidelines:

• Experimenters are unaware of (i.e., ‘blinded’) of the identity of samples or animal subjects which are anonymized whenever possible
• All experiments are carried out in at least three independent biological replicates (not just pseudo-replicates)
• All major conclusions are based on at least two experimentally distinct methods
• Biological sex is considered an independent biological variable whenever appropriate
• All experiments are documented in virtual notebooks stored in Stanford box
• All primary data are reviewed in lab meetings by multiple lab members, not just the P.I.

With these rules, we aim to promote transparency and reproducibility. Arguably the biggest challenge in maintaining science integrity at present is not the fabrication of data, but issues in analyzing and interpreting data. Unrestricted access to raw data will greatly help to meet this challenge because it enables independent and possibly alternative evaluations of the data. It is inevitable that our analyses and interpretations will be challenged. Gratifyingly these challenges will concern the substance of the science we publish instead of nitpicking and will facilitate more interactive scientific practices.

For our efforts in promoting data availability and transparency, our lab recently received the Stanford University Libraries Data Sharing Prize, an Open Science Award from CORES.

Social Media (PubPeer) Scrutiny of Südhof Lab Work

Websites such as PubPeer provide a potentially valuable service to the scientific community in identifying problems with published papers, generally focusing on discrepancies in a paper’s data but not on data reproducibility or conclusions. These posts have helped us to identify problems we overlooked and to correct errors we made (see detailed accounting of PubPeer posts below).

PubPeer critics recently used new sophisticated computational tools to analyze >25,000 images in our papers. PubPeer posts identified multiple errors we made that were previously undetectable without the new software. In addition, they, but also levelled many unfounded accusations. The PubPeer posts focus on one or two details in a paper, generally in older images, not on the actual science.  

Regrettably, however, PubPeer and other social media areis non-transparent, censors responses, and uses anonymous commentators with continuously changing aliases. Many PubPeer comments appear to pursue an agenda unrelated to science integrity.  PubPeer posts frequently exhibit a fundamentalist attitude that insists that even an accidental duplication of a control image, undetectable to the naked eye, is a major issue, demanding that science should be absolutely pure. Historically, representative images were meant to illustrate data for a better understanding of experiments and were not required to be ‘pure’. Splicing gel bands or changing images for illustrations was commonly accepted. A common PubPeer strategy is to repeat the same allegations in different ways to enhance impact, often accompanied by ‘animations’ that confer a veneer of seriousness. Even if an accusation is rather implausible, this strategy creates an aura of ‘something is wrong here’ that is difficult to counter. Thereafter, PubPeer posts are publicized on X and other media and communicated to journals, journalists, and university administrators. We nevertheless respond to PubPeer comments because journals often accept accusations without examining their plausibility.

PubPeer critics recently used new sophisticated computational tools to analyze >25,000 images in our papers. PubPeer posts identified multiple errors we made that were previously undetectable without the new software, but also levelled many unfounded accusations. The PubPeer posts focus on one or two details in a paper, generally in older images, not on the actual science. They exhibit a fundamentalist attitude that demands that science should be absolutely pure. Thise attitude of many PubPeer posts is deeply unscientific. We (and other sScientists) are human and make mistakes, as discussed below.  and nNo scientific work is ever perfect or final. Historically, representative images were meant to illustrate data for a better understanding of experiments and were not required to be ‘pure’. Splicing gel bands or changing images for illustrations was commonly accepted. Now, every hint of an image change, often introduced to improve the communication of results, leads to accusations of fraud even if there is no doubt about the actual conclusions of a study. The ‘Ethics’ departments of journals love to pick this up. No wonder few young people want to become scientists!

Finally, many PubPeer commenters maintain commercial websites communicating their discoveries and have a conflict of interest. Based on PubPeer posts, publications on sites such as ‘ForBetterScience’ and ‘Spectrum’ provide ad hominem criticisms of me as a person that are impossible to refute. Commentators continuously publicize their PubPeer criticisms on ‘X’ (formerly Twitter, run by Elon Musk) and Thereafter, PubPeer posts are publicized on X and other media and communicated them to journals, journalists, and university administrators. Prominent commentators are invited to give paid presentations about science rigor and reproducibility based on their PubPeer posts and social media presence and receive ‘integrity’ prizes without evidence of scientific expertise. We nevertheless respond to PubPeer comments because journals often accept accusations without examining their plausibility and we have to try to prove our innocence decades after papers were published.

Opportunities vs. Pitfalls of Computational Error Searches in the Literature

Two principal types of issues in papers are detected by new AI-powered programs such as ‘ImageTwin’ or ‘ProofIG’ used on PubPeer. First, image or data duplications that could be intentional or accidental copy-paste errors (these commonly occur when the copy function on a keyboard fails and a previously copied image or data element is inserted twice). Second, image changes that are either artifacts of image processing and reproduction or intentional image manipulations. For both issues, each identified ‘problem’ could represent an acceptable practice (i.e., no problem), a minor error made by the scientists or the journal, or an instance of misconduct. Two criteria can distinguish accidental errors and artifacts from misconduct. First, intent. Minor errors and artifacts are accidental isolated instances, often in a large number of images or data in a paper. Misconduct, conversely, is the willful manipulation of data usually by a repeat offender. Second, impact. Minor errors and artifacts provide no benefit to a paper, i.e. they do not improve a paper, whereas misconduct makes a paper look better. The wide availability of the new AI-powered programs that enable detection of these issues will prevent errors in future but will also, regrettably, facilitate creating fake images.

AI-powered searches for issues in papers, such as those performed by PubPeer commenters, have severe limitations. For example, when immunoblots are imaged at high resolution but reproduced at low resolution, the bands can assume a ‘halo’. The halo is not an intentional insertion of bands but simply represents a digital reproduction artifact. Similarly, immunoblots produced by the same gel apparatus with similar samples and analyzed with comparable secondary antibodies often exhibit the same shapes and features even if they are from different gels (see image below that illustrates the same artifact in four different blots with different samples!). Many false allegations of fraud on PubPeer suggest data duplications that simply don’t exist.

As another example, image analysis programs also detect image aberrations that are produced by the publishing process. In a widely publicized insinuation of fraud against my lab it was alleged that an immunoblot we published in 2010 (Supplementary Figure S6b of O.H. Shin et al.; # 37 below) was intentionally manipulated. This accusation was based on the following analysis:

The areas boxed in different colors were alleged to be intentionally duplicated. The boxed duplicated areas partly overlap, do not correspond to gel lanes or bands, and are randomly distributed. It would make no sense for a fraudster to duplicate such small areas of background – a fraudster could just run a gel with empty lanes – and such duplications do not improve the data. They are typical image reproduction artifacts. Similar artifacts in immunocytochemistry images have caused waves of criticisms. An accusation against another lab provides a beautiful example of how such artifacts occur (https://pubpeer.com/publications/C9E4F18C603C449A0CD32876B719A5). In the same vein, we could recently prove that a recent accusation against our lab of an image manipulation was false because, luckily and serendipitously, the first author retained the original blots (case #10 below). Importantly, it is even possible that the two image aberrations that we found in our retracted Neuron paper (case # 27 below) may represent such publication artifacts but because the raw data have been lost, we cannot prove our innocence here – in the world of science integrity ‘ethics’ one is guilty if one can’t prove one’s innocence no matter how old the data. A general problem is that digital reproductions of images can create artifactual duplications especially if the image resolution is changed during the publication process or if the images were obtained with older software. These artifacts are not visible to the naked eye but are now picked up by PubPeer accusers using sophisticated new software.

Source of errors in papers from the Südhof lab

As described below, more than 30 students and postdocs in my lab over 20 years acknowledged copy-paste mistakes in their papers. The presence of these copy-paste errors in many of our papers constitutes a systemic problem. Nearly all of these errors involve papers containing many (up to 500) images. Most errors concern control conditions where images look alike. Once a copy-paste mistake was committed, neither the student/postdoc who assembled the figure nor I could detect it until new computational image analysis became available. The nature of these mistakes – insertion of incorrectly duplicated representative images or duplicated numbers in data files after analysis – means that the mistakes have no effect on the findings or conclusions of a paper.

A plot of papers published (green circles, see below) and papers with errors (red circles) vs. the year of publication illustrates the temporal occurrence of our lab’s errors compared to the number of papers published.

This plot reveals that errors do not correlate with the number of papers published, lab size, or particular postdoctoral fellows/students in the lab, but coincide with the advent of pasting digital images or of publishing data excel files that are susceptible to copy-paste errors. A preliminary sampling of the literature revealed that similar copy-paste mistakes are ubiquitous in papers with lots of data. Indeed, many accusations against my lab actually identified copy-paste errors in collaborating labs. The software that enabled recent detection of these errors will also prevent their recurrence in future, together with the advances in raw data storage that are now in place.

In addition to the many copy-paste errors in our lab, reanalysis of the raw data of one paper (case #8 below) revealed that the raw data were incorrectly analyzed and that this mistake was compounded by copy-paste errors. These scientific errors (not identified by PubPeer) led us to retract the paper although the raw data are available, there is no evidence of data manipulations, and we continue to believe in the conclusions. Furthermore, we recently discovered with the help of an external image analysis expert that two representative images in another paper (case #27 below) contain inexplicable abnormalities, which again warranted a retraction of the paper even though the quantitative analyses and conclusions are correct and the results will be republished since the raw data for other parts of the paper are available.

Main PubPeer accusers of the Südhof lab

Originally most PubPeer contributors were idealistic science enthusiasts. More recently, professional “science integrity investigators” who may derive income from their activities seem to dominate. The criticisms increasingly imply that minor mistakes are not just human error but signify a bigger problem. The majority of accusation against our lab on PubPeer are made by four commentators, Dr. Elisabeth Bik, Dr. Maarten van Kampen (pseudonym Orchestus quercus), Patrick Kevin (pseudonym Actinopolyspora biskrensis), and Dr. Kaveh Bazargan (pseudonym Illex illecebrosus). The four major accusers of our lab often operate like a tag team creating an echo chamber in which one person (usually Dr. Bik) first makes an accusation and the others then repeat the same accusation multiple times from various angles.

Dr. Elisabeth Bik is a professional integrity consultant who may provide paid services to universities, journals and other customers. Dr. Bik specializes in image duplications and manipulations but appears to have no formal training in computational image analysis. Dr. Bik spent 15 years as a postdoctoral research associate at Stanford and several years in industry and science publishing before her current profession. Dr. Bik’s PubPeer posts on my lab are usually mirrored by tweets on Elon Musk’s ‘X’ platform implying misconduct without explaining the actual content of accusations or their relevance to the science. 

Dr. Maarten van Kampen frequently echoes Dr. Bik’s comments. He is a contributor to the website “ForBetterScience” run by Dr. Leonid Schneider that publishes ad hominem insults against me personally even though I have never met either him or Dr. Schneider. In repeating Dr. Bik’s accusations, Dr. van Kampen often uses probability arguments but appears to have no training in biomedical research.

Kevin Patrick also usually reinforces Dr. Bik’s accusations without new information. His comments often generalize individual mistakes to suggest systemic fraud. Like other accusers of the Südhof lab, Kevin Patrick collaborates with Dr. Leonid Schneider on the website “ForBetterScience” (see https://forbetterscience.com/2024/05/31/schneider-shorts-31-05-2024-historic-heavy-rain-and-flooding/#kevin) that publishes ad hominem attacks against me.

Dr. Kaveh Bazargan is a physicist with a PhD in display holography. He founded “River Valley Technologies” (https://rivervalley.io/), a company that offers paid publication and proofing services. Dr. Bazargan’s frequent comments on PubPeer on our work again generally repeat Dr. Bik’s accusations, mostly reiterating her allegations with ‘animations’ that provide no new information but are also amplified on ‘X’. He regularly asks for retractions based on single copy-paste mistakes.

The Economic and Personal Benefits and Costs of Scientific Fraud Detection Efforts

Fraud constitutes a potentially serious problem in science, especially when it is the basis for a large number of follow-up studies or clinical trials as described in a recent book (Charles Piller “Doctored: Fraud, Arrogance, and Tragedy in the Quest to Cure Alzheimer’s”). It is essential for scientific papers to present data that are accurate descriptions of experimental results even though no experiment is ever perfect. Papers with errors that affect their conclusions need to be retracted no matter whether or not these errors are unintentional (see case #8 below). Moreover, if a paper contains potentially manipulated data, even if it is just one figure among many, a retraction is generally considered necessary in the current fundamentalist climate of science. Such retractions are often necessary but can sometimes be questioned if the remaining data that are often obtained by scientists who had no part in the alleged manipulation are sound. Overall, continuous reevaluations of papers for mistakes are thus warranted.

Having said this, however, it should be noted that there is also a dark side to scientific fraud detection efforts. Often the identified mistakes are unintentional errors of no scientific consequence. The wide reporting of these mistakes on PubPeer, ‘X’, blogs and other social media causes anguish, ruins careers, and produces economic waste. Especially the costs to young scientists are horrendous. Accusations of minor errors often make junior scientists feel personally attacked. They have to be dealt with in extensive communications, record searches often dating back decades, corrections that are meaningless, and sometimes new experiments. In my own lab, such accusations have pushed multiple junior scientists – all female – out of experimental science and led to soul-searching among those who remained.

As scientists, we need to communicate to the public that we do make and acknowledge mistakes. But we also have to ask ourselves whether correcting decades-old minor mistakes that nobody would ever notice without A.I. software is warranted. Are we forced to conform to social media pressures from people who are uninterested in the actual science but claim that absolute purity in science is a justifiable goal? Moreover, if a paper contains data manipulations that constitute only a tiny fraction of a project, is it justified correct to retract the entire paper in the name of scientific purity, or shouldn’t we have a more appropriate mechanism to deal with this problem? These are issues that we, as a community, have to deal with.

Finally, PubPeer has guided journal practices beyond science. Journals have established ethics departments that investigate allegations of misconduct. The ‘ethics’ teams, however, have a conflict of interest because their existence depends on finding problemssomething, an inherent problem of regulatory bureaucracies. The independence of journal ethics teams from the editorial side of journals compounds the conflict of interest problem because the ethics teams may not be abledo not receive feedback on  to assess what is trivial and what has true implications for the science. As a result, minor infractions become amplified, leading to paper retractions where the problems are irrelevant for the findings of a paper. This creates waste, human suffering, and amplifies anti-science attitudes.

Complete Accounting of Südhof Lab Mistakes and Unfounded Allegations

Given the persistent public scrutiny of our lab's work, we here provide a full accounting of our lab's errors. We will discuss all PubPeer justified and unfounded accusations, describe our uncensored responses, and additionally reveal mistakes we identified that have not (yet) been listed on PubPeer.

Below, Südhof lab PubPeer accusations are summarized in reverse order of a paper's first criticism or of our identification of a problem. Accusations are numbered according to PubPeer entries, with gaps in numbers caused by responses or irrelevant comments. We classify accusations based on their resolution as ‘unfounded’, ‘minor error’, and ‘major error'. We include accusations directed against our lab but concerning data from collaborating labs. As scientists, we are often reluctant to accept mistakes. Realizing this tendency, we tried to concede errors wherever these exist, even if trivial. However, PubPeer accusers are also loath to admit mistakes in allegations and often repeat the same point, associated with fancy graphics and animations that purport to represent analyses. This frequently results in unending discussions on PubPeer in which our responses are censored, prompting us to provide this accounting for the sake of transparency. 

Total number of cases

• Sudhof papers with minor errors:                                           24
• Sudhof papers with major errors:                                            2
• Errors in papers by collaborators:                                            9
• Total number of unfounded accusations:                            68
• Number of accused Sudhof lab trainees:                             35
• Fraction of errors per image, graph, or table:            <0.002        

Detailed discussion of individual papers

49. Paper: Xu, J., Pang, Z.P., Shin, O.H., and Südhof, T.C. (2009) Synaptotagmin-1 functions as a Ca²⁺ sensor for spontaneous release. Nature Neurosci. 12, 759-766.

PubPeer Weblink: https://pubpeer.com/publications/FA163CBB31FF4A026B0E3DC0BF0365

#1 & #2  (January 2025)

Accusation: Two of the 174 electrophysiology traces among 174 traces in the paper look ‘unexpectedly similar’, implying they may be duplicated.

Resolution: The similarity in traces may be due to a copy-paste error, a serendipitous chance event, or an image duplication during the processing of the paper for publication. We no longer have access to the raw data that were archived at UT Southwestern, my former institution and cannot clarify this issue.

Classification: Unfounded or minor error during figure assembly or during reproduction

***

48. Paper: Fernandez-Chacon, R., Shin, O.-H., Königstorfer, A., Matos, M.F., Meyer, A.C., Garcia, J., Gerber, S.H., Rizo, J., Südhof, T.C., and Rosenmund, C. (2002) Structure/function analysis of Ca²⁺-binding to the C₂A-domain of synaptotagmin 1. J. Neurosci. 22, 8438-8446.

PubPeer Weblink: https://pubpeer.com/publications/1FD9572D003130580F6C598C39D9C6  

#1 & #2  (November 2024)

Accusation: Figure 7A exhibits an ‘irregular background’, implying it has been manipulated.  

Resolution: The irregular background is a typical artifact of older image processing and reproduction software. See the many other examples discussed below.

Classification: Unfounded

***

47. Paper: Hao, Y.A., Lee, S., Roth, R.H., Natale, S., Gomez, L., Taxidis, J., O’Neill, P.S., Villette, V., Bradley, Jl, Wang, Z., Jiang, D., Zhang, G., Sheng, M., Lu, D., Boyden, E., Delvendahl, I., Golshani, P., Wernig, M., Feldman, D.F., Ji, N., Ding, J., Südhof, T.C., Clandinin, T.R., and Lin, M. (2024) A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events. Neuron 112, 3680-3696.

PubPeer Weblink: https://pubpeer.com/publications/B86E470B8AF2DBE88B7956BCF08AF3

#1 & #2  (November 2024)

Accusation: One of the traces in a collaborator’s paper may be duplicated. 

Resolution: The collaborator confirmed that his postdoc duplicated the trace and corrected it.

Classification: Mistake by another lab

***

46. Paper: Pak, C.H., Danko, T., Zhang, Y., Aoto, J., Anderson, G., Maxeiner, S., Yi, F., Wernig, M., and Südhof, T.C. (2015) Human Neuropsychiatric Disease Modeling Using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1. Cell Stem Cell 17, 316-328.

PubPeer Weblink:  https://pubpeer.com/publications/4BF247658B9C771C11277691085AE5

#1 (July 2024)

Accusation: Two panels in Figure S6 are switched.

Resolution: Accidental copy-paste mistake during figure assembly

Classification: Minor error

#4, #6 & #7 (October 2024)

Accusation: The resting potential of the neurons analyzed in the paper is too low, and our explanation is false that such a low resting potential is commonly observed in human neurons derived from stem cells.

Resolution: Neurons derived from human stem cells mature slowly and exhibit a steady increase in resting potential during culture. In this particular paper, neurons were analyzed after 3 weeks in culture and are thus relatively immature. The -38 mV resting potential is not materially different from that observed in older neurons analyzed by others at 5-8 weeks. For example, Shih et al. (Stem Cell Res. 2021) observe -42-48 mV resting potentials after 4 weeks in culture and Chen et al. (Stem Cell Res. 2020) observe -50 mV after 12 weeks in culture. Even one of the examples mentioned by the accusers (Buhlmann et al., J. Neurosci. 2024) sees -45-50 mV resting potentials after 4 weeks, which isn’t very different from ours.

Classification: Unfounded

***

45. Paper: Um, J.W., Pramanik, G., Ko, J.S., Song, M.Y., Lee, D., Kim, H., Park, K.S., Südhof, T.C., Tabuchi, K., and Ko, J. (2014) Calsyntenins Function as Synaptogenic Adhesion Molecules in Concert with Neurexins. Cell Reports 6, 1096-1109.

PubPeer Weblink: https://pubpeer.com/publications/10E74576C4033F2A67749B3395FEFD

#1 (July 2024)

Accusation: Figure 3A contains an image duplication

Resolution: Our lab made only a minor contribution to this paper but the contributing lab who made this mistake acknowledged the duplication.

Classification: Minor error by another lab

***

44. Paper: Boucard, A., Maxeiner, S., and Südhof, T.C. (2014) Latrophilins Function as Heterophilic Cell-Adhesion Molecules by Binding to Teneurins: Regulation by Alternative Splicing. J. Biol. Chem. 289, 387-402.

PubPeer Weblink: https://pubpeer.com/publications/7B45E6171BCB1F3B805C2BDC316DF0

#1-4, #6 & #8 (July 2024)

Accusation: Figure 1G contains an image duplication

Resolution: Copy-paste error in the panels of Figure 1G

Classification: Minor error

***

43. Paper: Arancillo, M., Min, S.W., Gerber, S., Münster-Wandowski, A., Wu, Y.J., Herman, M., Trimbuch, T., Rah, J.C., Ahnert-Hiler, G., Riedel, D., Südhof, T.C., and Rosenmund, C. (2013) Titration of Syntaxin 1 in mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability. J. Neurosci. 33, 16698-16714.

PubPeer Weblink: https://pubpeer.com/publications/25105898138827EDD8E4A7332B871D

#1 & #2

Accusation: The Rab3 and NSF blots in Figure 1G are called ‘remarkably similar’ and the blots were put together from different gels.

Resolution: High-magnification images show that the blots are clearly different. Although the bands are similar as expected for blots run on the same gel apparatuses, the lanes ran differently, with a much larger gap between lanes for the smaller Rab3A protein than the larger NSF protein, again as expected (see red circles in image). Moreover, assembling illustrative blots from different lanes was perfectly acceptable practice in science before PubPeer started to criticize it. The blot predates the digital era and was obtained using ECL and film at UT Southwestern.

Classification: Unfounded

***

42. Paper: Sun, W., Liu, Z., Jiang, X., Chen, M.B., Dong, H., Liu, J., Südhof, T.C., and Quake, S. R. (2024) Spatial and single-cell transcriptomics reveal neuron-astrocyte interplay in long-term memory. Nature 627, 374-381.

PubPeer Weblink: https://pubpeer.com/publications/6123C544EAE3A2F1FE1D9CE476E00C

#1 (July 2024)

Accusation: None – simply cites an unreviewed preprint that alleges that our study is ‘unlikely to replicate in future’ because the authors of that preprint did not agree with some of the statistical analyses in our paper.

Resolution: It is impossible to respond to a reference to an unpublished paper, except to say that this post obviously ‘questions’ yet another of our papers to add to the success list of PubPeer claiming to have identified ‘questionable’ papers from our lab. However, we applaud the BioRxiv paper authors for initiating an open scientific discussion in which we will engage instead of the anonymous PubPeer allegations that are impossible to discuss fairly.

Classification: Uninterpretable accusation

Postscriptum: A scientific discussion on the issues raised by the preprint mentioned by the accuser will be published

***

41. Paper: Gallardo, G., Schlüter, O.M., and Südhof, T.C. (2008) A molecular pathway of neurodegeneration linking a-synuclein to ApoE and Ab-peptides. Nature Neurosci. 11, 301-308.

PubPeer Weblink: https://pubpeer.com/publications/02B530E2D617B034C0E76407D16B99

#1 & #3 (July 2024)

Accusation: A blot duplication exists in Figures 2C and 6C.

Resolution: High magnification images show that the two blots exhibit distinct backgrounds, showing they are different.

Classification: unfounded

#4 (July 2024)

Accusation: The error bars are missing in Figure 4a.

Resolution: The error bars are not missing. The SEMs were simply too small to be visible in the graph.

Classification: unfounded

***

40. Paper: Shin, O.-K., Rizo, J., and Südhof, T.C. (2002) Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells. Nature Neurosci. 5, 649-656.

PubPeer Weblink: https://pubpeer.com/publications/9BAEF55F4945697536FB528482F5B7

Date of first mistake allegation: May 2024

#1 to #6 (May 2024)

Accusation: The accusers allege that among the 70 different blots shown in this paper, bands on 8 blots look ‘surprisingly similar’, implying that the images were manipulated. In total, in pairwise comparisons 9 ‘surprising’ similarities were alleged among a total of 556 bands (>300,000 comparisons in total). All allegedly duplicated bands are adjacent on the same blots. Thus, the allegations here are not that blots were duplicated but that a fraction of adjacent bands were duplicated.

Resolution: Since the accusers somehow convinced the ‘ethics’ department of Nature Neuroscience that their allegations are worthy an investigation and since PubPeer does not enable a scientific discussion, we respond here in detail.

The blots reported in this paper were produced by ECL amplification of immunoreactive bands that were visualized on film and scanned or photographed (not sure which since the blots were obtained 25 years ago). Photographs of the blots were then pasted manually onto a paper, annotated with Letraset, and again photographed as a figure for a paper. The original raw data were archived as required by law at UT Southwestern when I left that institution in 2008.

High-magnification images of 3 examples of blots alleged to represent duplications among reveal four interesting features:

1. The bands are indeed similar – only 9 such cases exist out of >300,000 comparisons.
2. The 9 similar bands are ALWAYS next to each other.
3. The background surrounding the similar bands contains many non-identical features (yellow circles) and is thus clearly different, i.e. not duplicated.
4. There is no sign of band ‘splicing’ or of any other manipulations of the images even though other bands in the blots are clearly spliced. These spliced bands were transparently moved from the right to the left of the blots for illustrative purposes at a less fundamentalist time when such ‘splicing’ was standard acceptable practice.

The fact that there are no indications of band splicing or other physical or digital manipulations is important given that these experiments were performed decades ago with non-digital methods. At that time, no AI-driven image reconstructions were available to seamlessly insert bands of proteins into different backgrounds. The fact that other bands were transparently ‘spliced’ shows that such splicing is easily detectable.

How can we explain the fact that 9 out of 556 bands are similar but the images are overall non-identical and there is no sign of image manipulations? Two plausible explanations are possible. First, that this is an accidental similarity in <2% of the bands of similar samples run on the same gels. Second, that the image processing software during scanning of the blots or during publishing may have introduced duplications of internal image components when the software perceived these components to be the same, even if they are not the same. Such phenomena explained other alleged image manipulations on PubPeer. Digitization and compression algorithms, especially older ones, used ‘pattern matching’ and economize by simply duplicating matched patterns. A similar error was observed in old Xerox copy machines “The source of the error was a bug in the JBIG2 implementation, which is an image compression standard that makes use of pattern matching to encode identical characters only once. While this provides a high level of compression, it is susceptible to errors in identifying similar characters.” (see https://en.m.wikipedia.org/wiki/Xerox#Character_substitution_bug). This has also been documented for immunocytochemistry images in older papers (see https://pubpeer.com/publications/C9E4F18C603C449A0CD32876B719A5, an example where the responsible scientist was lucky to retain decades old images). We are not image analysis algorithm experts and cannot assess what older image processing and reproduction software may or may not have done to images, nor do we know why only some bands are ‘pattern matched’ whereas others are not.

Classification: unfounded

#8, #9 & #11 (July 2024)

Accusation: Various commenters claim that it is impossible that the isolated band similarities are due to a pattern matching algorithm artefact or a similar computational error in image processing downstream of the actual data production.

Resolution: We are not sure whether the commenters have the computer science background to make such a definitive judgement about image processing and reproduction software in use 25 years ago, but we certainly don’t have that expertise. Moreover, we are disturbed that we are thought guilty if we cannot prove our innocence. The commenters/accusers do not actually provide an explanation for the small fraction of similar adjacent bands or why the bands are similar yet clearly different. Instead, the commenters demand that we explain the similarities and not the differences. If we can’t, we are damned.

Furthermore, the commenters claimed that the images could have been digitally manipulated at the time of the submission of the paper 25 years ago because software at that time, according to the commenters, was supposed to have been available (again, we don’t know if this is true). However, at the time at which this paper was submitted we did not use digital procedures to process and submit figures. Figures were submitted in physical paper form. Nature Neuroscience did not have an on-line submission system until 2002. Figures were simply mounted on a paper and photographed, and the photos were submitted.

Classification: unfounded

#12-14 (September 2024)

Accusation: Additional commenters now claim to detect signs of splicing in one of the allegedly manipulated bands as a sharp edge.

Resolution: In their zeal to find fault with our work, our accusers here allege band splicing because of a sharp edge at some of the bands reproduced at low resolution with large pixels. Importantly, the accusations doesn’t mention that in this paper we transparently spliced together many bands. See the example from the figure attacked by the commenters (arrows identify obvious lane splicing, box shows the bands criticized by our accusers). Splice sites are obvious without an attempt at deception:

At the time of this paper’s publication, band splicing was a common acceptable practice and we made no attempt to hide it. Band splicing was done manually by cutting out bands and pasting them on paper (there was no electronic submission or electronic figure preparation).  In some panels, all lanes are ‘spliced’. The allegation that a postdoc openly spliced bands together at one position in a figure but tried to hide the splicing at another position beggars belief.

Of course our accusers will now say that they don’t speculate on motivation and that they only state facts but that is not true. PubPeer commenters select features of a figure to sow doubts about it but leave out other features that are relevant and state as facts speculations without an assessment of the science. Throughout the hundreds of accusations against our lab, our accusers always claim that they can judge which image processing and image reproduction software artifacts are ‘suspicious’ without real evidence – they have always been proven wrong when old data were actually available.

Classification: unfounded

***

39. Paper: Hosaka, M., Hammer, R.E., and Südhof, T.C. (1999) A phospho-switch controls the dynamic association of synapsins with synaptic vesicles. Neuron 24, 377-387.

PubPeer Weblink: https://pubpeer.com/publications/3A1A9BCBA901F9A66ED727A7912877

Date of first mistake allegation: June 2024

#1 (June 2024)

Accusation: The immunoblots for Synaptogyrin and Rab3a in Figure 8 look ‘remarkably similar’, implying that they are duplicated.

Resolution: This is a typical A.I.-driven erroneous identification of a non-existing blot duplication. The two blots are expected to be 'remarkably similar' given that Synaptogyrin and Rab3a are both synaptic vesicle proteins which biologically behave the same way in this particular experiment, and that the immunoblots are for proteins of similar but different molecular weights (~35 kDa vs. ~25 kDa) that were analyzed on the same gel and probed on the same blot which was cut into stripes. As a result, the bands exhibit the same relative changes and display the same blotting artifacts. Note also that this blot derives from the predigital area when copy-paste mistakes were very unusual.

Classification: unfounded

#2

Accusation: It is improper for us to thank a lab member for an initial technical contribution to the project in the Acknowledgement section of the paper.

Resolution: We do not believe that thanking a lab member in an Acknowledgement section is inappropriate.

Classification: unfounded

#4, #6 #8, #10, #12 & #14 (July 2024)

Accusation: Further accusations alleging, without new information, that the two blots are the same and that there is a suspicious duplication.

Resolution: To avoid the real possibility that a journal, Stanford, or NIH takes these fraud allegations seriously, I will describe in detail these experiments that were performed nearly 30 years ago. The blots reported in this paper were produced by ECL amplification of immunoreactive bands that were visualized on film and then scanned or photographed. The scanned or photographed images were then manually pasted into an illustration on paper, labeled using Letraset, photographed, and submitted for publication as a photograph since there was no digital submission system. The original raw data were archived as required by law at UT Southwestern when I left that institution in 2008. A high-magnification image of a section of the blot is shown below:

The bands are indeed very similar as would be expected from the experiment (see explanation above). In fact, it would be worrisome if they weren’t. However, there are significant differences that show that the blots are NOT identical (see areas marked by yellow circles that exhibit differences in background). These subtle differences in addition to the fact that the bands exhibit distinct thicknesses as would be expected again from slightly different antibody affinities show that the blots are different.

Classification: unfounded

#16 (July 2024)

Accusation: The ‘moderator’ (who may well be identical to the major accusers of our lab) states “Dr Südhof is obviously unwilling and/or unable to address the issues. However unsatisfactory that conclusion may be, we may limit fruitless, repetitive discussion.”, basically claiming that our responses do not address the issues.

Resolution: We feel this comment exposes the nature of PubPeer: Even the ‘moderator’ is partisan. No wonder that our comments get censored. PubPeer reveals itself as being an anti-science ‘X’ like platform with opaque funding and intransparent backers.

Classification: unfounded

***

38. Paper: Pang, Z.P., Sun, J., Rizo, J., Maximov, A., and Südhof, T.C. (2006) Genetic Analysis of Synaptotagmin 2 in Spontaneous and Ca2+-Triggered Neurotransmitter Release. EMBO J. 25, 2039-2050.

PubPeer Weblink: Not yet publicized by PubPeer but posted on the first author’s website

Date of first mistake identification/allegation: May 2024

#1

Mistake identified: Supplementary Figure S1 contains a single blot image duplication involving a control condition.

Resolution: The journal has been contacted and the correct blot has been submitted.

Classification: Minor error identified by lab

***

37. Paper: Shin, O.-H., Lu, J., Rhee, J.-S., Tomchick, D.R., Pang, Z.P., Wojcik, S., Camacho-Perez, M., Brose, N., Machius, M., Rizo, J., Rosenmund, C., and Südhof, T.C. (2010) Munc13 C2B-domain – an activity-dependent Ca2+-regulator of synaptic exocytosis. Nature Struct. Mol. Biol. 17, 280-288.

PubPeer Weblink: https://pubpeer.com/publications/CF9BF3F6AF7EACB2FB3C0581A8AB76

Date of mistake identification/allegation: May 2024

#1 and following comments (May 2024)

Accusation: The  immunoblot stripes in supplementary Figure S6b (reproduced digitally at low resolution by the journal from a non-digital original blot) contains tiny areas of microduplications in the background pattern (not the actual signal).

Resolution: This was originally an unusual accusation that has since been repeated for other very old blots and images from our lab. The accusation targets bits and pieces of two blot stripes from the pre-digital era in a supplementary figure panel containing 7 such stripes. If these micro-areas were truly intentionally duplicated and not created by an image-processing artifact with a non-digitally acquired blot that is reproduced at low resolution, then these manipulations would serve no purpose since they are in an area of the blot that contains no signal. Moreover, the allegedly intentionally duplicated micro-areas do not conform to the lanes of the blot or to the protein signal. Finally, the two sets of allegedly duplicated micro-areas are only within a blot stripe but not between blot stripes (this interestingly resembles case #40 above). The last finding is revealing: clearly the scrambling of bits of image pieces was intrinsic to the image.

The accusers could argue that maybe there were lots of smudges on the gel that were supposed to be covered up, with each smudge covered by a different ‘cloned’ micro-area. However, there is no evidence of smudges in any of the blots. Moreover, the covering would have to be done without a trace at a time at which seamless image manipulations were difficult and would use different pieces within a blot without sharing between blots – a scenario that is highly unlikely. Furthermore, all manipulations would have to be performed on physical blots pasted onto a paper. The most likely explanation for this finding, like for many of the ‘mistakes’ identified by A.I.-powered software, is that these random microduplications are simply a reproduction artifact of a digitized image derived from a photograph.

Classification: unfounded

***

36. Paper: Maximov, A., Shin, O.-H., and Südhof, T.C. (2007) Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release. J. Cell Biol. 176, 113-124.

PubPeer Weblink: https://pubpeer.com/publications/1623AD50C70F6D92C1BD05B69EEED3

Date of mistake identification/allegation: May 2024

#1 & #2 (May 2024)

Mistake identified: A control electrophysiology trace may be duplicated.

Resolution: We will contact UT Southwestern which retained all original data by law to determine if this is a duplication or simply a similar trace.

Classification: Likely minor error or a serendipitously similar trace

#4 (August 2024)

Mistake identified: The accuser demands to see the original traces

Resolution: As mentioned above, the experiments were performed 20 years ago at UT Southwestern, which retained all original data as required by law. These data are property of UT Southwestern and no longer under our control.

Classification: Irrelevant comment

***

35. Paper: Gokce, O., and Südhof, T.C. (2013) Membrane-Tethered Monomeric Neurexin LNS-Domain Triggers Synapse Formation. J. Neurosci. 33, 14617-14628. PMCID: PMC3761060

PubPeer Weblink: https://pubpeer.com/publications/DB2D4970F91BF62729727D5DE39975

Date of mistake identification/allegation: We identified and corrected this mistake in the journal but Dr. Bik afterwards repeated our own identification of the mistake without new information

#1 (May 2024)

Mistake identified: The paper contains a copy-paste mistake

Resolution: No resolution necessary. A correction has been published

Classification: Minor mistake uncovered by the lab

***

34. Paper: Lee, K., Kim, Y., Lee, S.-J., Qiang, Y., Lee, D., Woo Lee, H., Kim, H., Je, H.S., Südhof, T.C., and Ko, J. (2013) MDGAs selectively interact with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development. Proc. Natl. Acad. Sci. U.S.A. 110, 336-341.

PubPeer Weblink: Not yet publicized by PubPeer but PNAS published a Correction

#1 (May 2024)

Mistake identified: Image duplication in Suppl. Figure 2B

Resolution: A correction has been published by the journal

Classification: Minor mistake by another lab and uncovered by the lab

***

33. Paper: Wöhr, M., Fong WM, Janas JA, Mall M, Thome C, Vangipuram M, Meng L, Südhof, T.C., and Wernig, M. (2022) Myt1l haploinsufficiency leads to obesity and multifaceted behavioral alterations in mice. Mol. Autism, 13, 19. PMCID: PMC9087967

PubPeer Weblink: https://pubpeer.com/publications/6AEE2ED764E4EA67E352B62B92D882

#1 (April 2024)

Accusation: Image duplication in Supplementary/Additional File 2

Resolution: The accusation is likely correct but the data are from a collaborating lab

Classification: Likely minor mistake from another lab

***

32. Paper: Ko, J., Soler-Llavina, G.J., Fuccillo, M.V., Malenka, R.C., and Südhof, T.C. (2011) Neuroligins/LRRTMs prevent activity- and Ca²⁺/calmodulin-dependent synapse elimination in cultured neurons. J. Cell Biol. 194, 323-334.

PubPeer Weblink: https://pubpeer.com/publications/6A24D71A02F213024FBA3452BD89DA

#1  (April 2024)

Mistake identified: Supplementary Figure 3 contains a duplicated image

Resolution: We self-reported the error

Classification: Minor mistake identified by us

***

31. Paper: Ko, J., Fuccillo, M., Malenka, R.C., and Südhof, T.C. (2009) LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation. Neuron 64, 791-798.

PubPeer Weblink: https://pubpeer.com/publications/13DFE3B5D880F57C51C7ACB03CFC6C

#1 (April 2024)

Mistake identified: Supplementary Figure 3 contains a duplicated image

Resolution: We reported the error

Classification: Minor mistake identified by us

#2 (April 2024)

Accusation: Supplementary Figure 3 contains a second duplicated image

Resolution: We missed this mistake when we reported the first error but confirm that the second mistake exists in the same figure

Classification: Minor mistake

#4 (July 2024)

Accusation: Repeat of accusation #2 three months after it was resolved

Resolution: No need for additional resolutions

Classification: Irrelevant comment

***

30. Paper: Shimojo, M., Madara, J., Pankow, S., Liu, X., Yates, J. 3rd, Südhof, T.C., and Maximov, A. (2019) Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity. Genes and Dev. 33, 365-376.

PubPeer Weblink: https://pubpeer.com/publications/2999ACA0C61FDB9B3F71E366688A37

#1 & #3 (April 2024)

Accusation: In Figure 1D some immunoblot sections containing no signal may be duplicated.

Resolution: The figure and data were not from the Südhof lab, but the senior author responded on PubPeer to explain that this allegation is unfounded

Classification: Unfounded

#2 (April 2024)

Accusation: Dr. Bik claims that since I am the only author from my lab on this paper, I must be an honorary author which is improper

Resolution: My affiliation on the paper was listed as UT Southwestern, as was that of the senior author Dr. Maximov. Dr. Maximov initiated the project in my lab at UT Southwestern and took the project and the reagents he generated with him in 2007 to his own lab, where he completed the project.

Classification: Unfounded

#4 (April 2024)

Accusation: The blots shown in Supplementary Figure 1 are overexposed

Resolution: This is a question of taste. These blots were from the pre-digital age when we often preferred long exposures with ECL visualizations to identify possible minor signals.

Classification: Unfounded

***

29. Paper: Sando, R., Jiang, X., and Südhof, T.C. (2019) Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins. Science 363, pii: eaav7969.

PubPeer Weblink: https://pubpeer.com/publications/3782092ABD5E5AC83CBB9F899C0D59

#1, #2, #4 & #6 (April 2024)

Accusation: Figure 3B contains image duplications.

Resolution: The image duplications produced by a copy-paste mistake were undetectable without specialized software but have now been corrected.

Classification: Minor mistake

***

28. Paper: Eichel, K., Uenaka, T., Belapurkar, V., Lu, R., Cheng, S., Pak, J.S., Taylor, C.A., Südhof T.C., Malenka, R., Wernig, M., Özkan, E., Perrais, D., and Shen, K. (2022) Endocytosis in the axon initial segment maintains neuronal polarity. Nature 609, 128-135.

PubPeer Weblink: https://pubpeer.com/publications/5423F032DB2E73F1ACD449E4B5BA96

#1 & #2 (April 2024)

Accusation: Extended Data Figure 5a contains a blot image duplication

Resolution: The allegations is likely correct but the data are not from a our lab

Classification: Minor mistake in a collaborator’s paper

***

27. Paper: Chen, L.Y., Jiang, M., Zhang, B., Gokce, O., and Südhof, T.C. (2017) Conditional Deletion of All Neurexins Defines Diversity of Essential Synaptic Organizer Functions for Neurexins. Neuron 94, 611-625.

PubPeer Weblink: A copy-paste mistake was initially discovered by the lab and later amplified by PubPeer https://pubpeer.com/publications/F7C42C356B2E7049FDB68A434EF4F8. However, Dr. Matthew Schrag subsequently discovered more serious image problems

#1 & #2 (April 2024)

Mistake identified: The paper’s first author, Dr. L.Y. Chen, reported that she inadvertently and incorrectly copy-pasted an image in Figure 2D into Figure S3A, creating an image duplication

Resolution: We reported this mistake on PubPeer as soon as we discovered it; a correction was filed.

Classification: Minor mistake identified by us.

#3-18 (April 2024)

Accusation: Figure S4B contain cloned micro-areas but an image analysis expert (Dr. M. Schrag) notes that such microareas are often image processing artifacts

Resolution:  As described, cloned micro-areas are often image processing artifacts. See another recent accusation by Dr. Bik that reveals how common such artifacts are (see https://pubpeer.com/publications/C9E4F18C603C449A0CD32876B719A5). The comments did not identify the real problem to which Dr. Schrag alerted us outside of PubPeer, namely that figure S4B contains a partial image duplication in the green channel.

Classification: The PubPeer accusations by themselves are unfounded but the figure contains problems not identified by PubPeer.

#25-67 (May to July 2024)

Accusation: In extensive repetitive posts, the four standard accusers of our lab and others claim that Figure S4B contains tiny subsections that appear to be duplicated within two images and are thus ‘cloned’, i.e. intentionally manipulated within that image. Moreover, the comments return to Figures 2D and S3A that the first author in post #1 reported to be a mistake. The accusations focus micro-duplications that are typical image processing artifacts that may have occurred during image stitching or article production.

Resolution: An intentional image manipulation involving micro-duplications makes no sense because it the effect is to make the image worse, not better. However, subsequent image analyses that were performed by an external expert discovered possible manipulations in Figure 1D that were not micro-duplications but involved the insertion of the same blue background vGluT2 control stain in both the test and control condition. This manipulation could have been intentional but its purpose is puzzling since it doesn’t achieve anything. Moreover, a second image also contains aberrations that are different from microduplications and that we cannot explain since the original images were lost. The discovery by the independent image analysis expert of a possible image manipulation has been referred to the journal and Stanford

Classification:  Pending.

Postscriptum: Based on our analysis with an invaluable contribution of an independent analysis performed by Dr. Matthew Schrag, we conclude that this paper has three problems, two minor and one major. First, an inadvertent image duplication error (post #1); second, duplications of micro-areas in cerebellar figures that are likely image processing artifacts (posts #3-67); third, inexplicable duplications of larger image areas in only one but not the other color channel in Figure 1D and S4B that cannot be readily accounted for by image processing artifacts. Neither the minor nor the major problem could be identified without artificial intelligence search software. Since the third problem cannot be explained as a copy-paste error and the raw data for these particular images in the paper cannot be located for further investigations, we are retracting the entire paper with the following notice: “We, the authors of this publication, have decided to retract the paper because we found that the images in figure 1D and Figure S4B contain aberrations that cannot be explained and the original data for these figures are missing. Raw data for the other components of the paper are available and their reanalysis confirmed the conclusions of the paper. We would like to thank M. Schrag for bringing these image aberrations to our attention.”

***

26. Paper: Seigneur, E., Polepalli, J., and Südhof, T.C. (2018) Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs. Proc. Natl. Acad. Sci. U.S.A. 115, E10235-E10244.

PubPeer Weblink: https://pubpeer.com/publications/25B0A673C668139C77CEAC19A99B11

#1 (April 2024)

Accusation: We discovered and reported two image duplications in Figure S4

Resolution: Posted by us to preempt social media shaming

Classification: Minor mistake identified by us.

#2 (July  2024)

Accusation: Our attempt to preempt a social media accusation because of this copy-paste mistake failed – the same mistake was repeatedly reported on PubPeer 3 months after it was reported

Resolution: None necessary

Classification: Irrelevant comment

***

25. Paper: Mall, M., Kareta, M.S., Chanda, S., Ahlenius, H., Perotti, N., Zhou, B., Grieder, S.D., Ge., X., Drake, S., Ang, D.E., Walker, B.M., Vierbuchen, T., Fuentes, D.R., Brennecke, P., Nitta, K.R., Jolma, A., Steinmetz, L.M., Taipale, J., Südhof, T.C., and Wernig, M. (2017) A proneuronal transcription factor repressing many non-neuronal fates. Nature 544, 245-249.

PubPeer Weblink: https://pubpeer.com/publications/D4D96DB690DAC5A3D912EE64E92AC3

#1 & #2 (April 2024)

Accusation: Extended Data Figure 5 contains two image duplications.

Resolution: The allegation concerns data that are not from the Südhof lab but are correct. The postdoc from the Wernig lab involved has initiated a Correction with the journal.

Classification: Minor mistake by another lab

***

24. Paper: Bacaj, T., Ahmad, M., Jurado, S., Malenka, R.C., and Südhof, T.C. (2015) Synaptic Function of Rab11Fip5: Selective Requirement for Hippocampal Long-Term Depression. J. Neurosci. 35, 7460-7474.

PubPeer Weblink: https://pubpeer.com/publications/52D37ED6C4D16682694521FA04B2BA

#1, 2, 4 & 6 (April 2024)

Accusation: In Figure 3D the immunoblotting bands for Cpx and Sph are alleged to look ‘unexpectedly similar’, implying they are duplicated.

Resolution: This allegation is based on a common image analysis mistake. Immunoblotting bands are expectedly similar when samples run on the same apparatus and analyzed by Coomassie or blotted with similarly clean secondary antibodies as described above. 

Classification: Unfounded

***

23. Paper: Golf, S,R,, Trotter, J,H,, Nakahara, G., and Südhof, T.C. (2023) Astrocytic Neuroligins Are Not Required for Synapse Formation or a Normal Astrocyte Cytoarchitecture. bioRxiv 10:2023.04.10.536254. doi: 10.1101/2023.04.10.536254. Preprint.

PubPeer Weblink: https://pubpeer.com/publications/98784D9AF9B1E8B5B1818E516B5001  

#1 (March 2024)

Accusation: One control panel among the 70 blots of Figure 3 is duplicated.

Resolution: There are two problems with this accusation. First, this is not a published paper, but a preprint posted to elicit comments on BioRxiv. The accuser chose not to comment on BioRxiv but to level an accusation on PubPeer which is typical of PubPeer’s anti-scientific agenda. Second, blot duplications are often mis-identified because similar samples, when run on the same gel apparatuses at the same relative positions in the gel produce the same blotting artifacts. This doesn’t mean they are duplicated. We have resubmitted the paper for publication and have made all raw data available.

Classification: Unfounded

#3 & #4 (April 2024)

Accusation: The accusers repeat the allegation of #1.

Resolution: Same response as for the first allegation

Classification: Unfounded

#6 & #7 (April 2024)

Accusation: The accusers demand to see the unpublished raw data.

Resolution: We consider the demand for unpublished raw data inappropriate

Classification: Unfounded

***

22. Paper: Seigneur, E., and Südhof, T. C. (2018) Genetic ablation of all cerebellins reveals synapse organizer functions in multiple regions throughout the brain. J. Neurosci. 38, 4774-4790.

PubPeer Weblink: https://pubpeer.com/publications/D8EAA6F915EE4008B654738F66ABE6

#1-#3 (March 2024)

Accusation: A blot of the 72 blots in Figure 2 and 36 graphs and an image of the 67 images in Figure 3 are duplicated.

Resolution: The allegation is correct. Once we copy-pasted the wrong blots and images we would have been unable to detect it with A.I.-driven software. Original data are now posted on PubPeer.

Classification: Minor mistake

#8 (April 2024)

Accusation: The accusers demand a journal correction.

Resolution: A journal correction has already been made.

Classification: Irrelevant

#9 & #11, 12, 16 & 19 (April 2024)

Accusation: The accusers repeatedly claim that the fact that 25 postdocs in the Sudhof lab over 15 years committed individual copy-paste errors suggests that there is systemic fraud in the lab. Moreover, the accusers also warrant that they have no conflict of interest.

Resolution: No resolution possible although the blanket condemnation of our lab by the same group of people seems very personal. Accusing 25 postdocs of fraud for isolated copy-paste mistakes in a tiny fraction of their data appears excessive. Note that the raw data for this paper have already been posted previously (https://purl.stanford.edu/cc564dr1376).

Classification: Irrelevant but quite revealing of the accusers’ motivations

#23 (July 2024)

Accusation: another repetition of the same accusations.

Resolution: No resolution necessary.

Classification: Pointless repetition of a resolved accusation

***

21. Paper: Wang, J., Miao, Y., Wicklein, R., Sun, Z., Wang, J., Jude, K.M., Fernandes, R.A., Merrill, S.A., Wernig, M., Garcia, K.C., and Südhof, T.C. (2021) RTN4/NoGo-Receptor Binding to BAI Adhesion-GPCRs Regulates Neuronal Development. Cell 184, 5869-5885. PMCID: PMC8620742

PubPeer Weblink: https://pubpeer.com/publications/5813077CE8B5C29E479FD50C259F77

#1 (March 2024)

Accusation: A supplementary figure containing 36 panels of illustrative images in a paper with 373 images and more than 130 graphs contains a single duplication of a control image

Resolution: The accusation is correct

Classification: Minor mistake

#6, 8, 10, 12, 14 & 15  (July 2024)

Accusation: Repetition of the same accusation with ‘animations’

Resolution: Same as above

Classification: No need for a response except to note that the enthusiasm in repeating allegations is interesting – PubPeer is a huge echo chamber

***

20. Papers: Jiang, X., Sando, R., and Südhof, T.C. (2021) Multiple signaling pathways are essential for synapse formation induced by synaptic adhesion molecule. Proc. Natl. Acad. Sci. U.S.A. 118, e2000173118; Li, J., Xie, Y., Cornelius, S., Jiang, X., Sando, R., Kordon, S., Pan, M., Leon, K., Südhof, T.C., Zhao, M., and Araç, D. (2020) Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism. Nature Comm. 11, 2140.

PubPeer Weblink: https://pubpeer.com/publications/027E93962D3C5DB86482283739C67D#10

#9 (March 2024)

Accusation: The two papers cited above use the same control image even though the stated conditions appear different, suggesting fraud

Resolution: The same control image was indeed used for the same type of experiment in two different studies performed at the same time. In the Nature Communications paper the control condition is labeled as 'Ctrl' whereas in the PNAS paper it is labeled as 'NPR-mut', which may seem to indicate that different conditions were attributed to the same images in two different papers. However, the Methods section clearly states that the 'Ctrl' of the Nature Communications paper is the 'NPR-Mut' that was used in the PNAS paper and thus the conditions are the same. It would have been better if we had indicated that the same control condition was used for the same experiment in two different projects, but this is perfectly proper since the experiments were done in parallel simultaneously. Five years ago we lived in a less prosecutorial environment and the lead author did not think of stating this explicitly, which was an oversight.

Classification: Unfounded

***

19. Paper: Burré, J., Sharma, M., and Südhof, T.C. (2012). Systematic Mutagenesis of a-Synuclein Reveals Distinct Sequence Requirements for Physiological and Pathological Activities. J. Neurosci. 32, 15227-15242.

PubPeer Weblink: https://pubpeer.com/publications/0FECC6D2E9498F9876CFCC24D2E03E#8

#1-#3 (December 2023 & March 2024)

Accusations: The blots shown in Figure 5, 6 and 7 are inappropriately assembled from multiple individual blots.

Resolution: These blots combine analyses of 26 samples that cannot be examined on single gels. Thus, the samples were run in parallel on multiple gel electrophoresis apparatuses and blots. No attempt was made to hide this fact. A decade ago, before the current atmosphere of prosecution, composite blots like the one cited by the accusers that are assembled from multiple individual blots were acceptable common practice. Nowadays this is labeled deceptive even though there is no attempt to hide it.

Classification: Unfounded

#4 - #6 (March 2024)

Accusations: Presumably based on artificial intelligence searches, the accuser identified image duplications in two sets of panels in Figure 7D and in Figure 9A.

Resolution: The accusation is correct. During copy-pasting of representative images, images were erroneously duplicated and differentially cropped. This mistake likely happened during copy-pasting of representative images and was undetectable before A.I. tools became available.

Classification: Minor mistake

#9 (March 2024)

Accusations: The accuser repeats the accusation that the combination of different blots in Figures 5 and 6 is inappropriate because different blots were combined at different positions.

Resolution: We disagree. At the time of this study -15 years ago- people found this perfectly acceptable.

Classification: Unfounded

#16 & #18 (April 2024)

Accusations: The accuser alleges a pattern of fraud in my lab because we retracted another paper in which we (not PubPeer) identified an inappropriate analysis of data. The comment implies that Dr. Burre’s isolated errors are also fraud and that more than 25 members of my lab each committed fraud by copy-paste errors in their papers.

Resolution: It is unfortunate that PubPeer has become a forum for people who appear to have an anti-science agenda expressed with implausible suspicions and insinuations. The occurrence of multiple isolated copy-paste errors in our papers that could only be identified using AI-driven software and that likely similarly exist in hundreds of other papers is not an indication that each of the responsible postdocs committed fraud.

Classification: Inappropriate comment.

#19 (July 2024)

Accusations: The accuser provides a belated animation of comment #4 that was already resolved, possibly to amplify the accusation even though it was long dealt with.

Resolution: No need for further responses.

Classification: Unfounded

***

18. Paper: Lin, P.Y., Chen, L.Y., Jiang, M., Trotter, J.H., Seigneur, E., and Südhof, T.C. (2023) Neurexin-2: An Inhibitory Neurexin That Restricts Excitatory Synapse Formation in the Hippocampus. Sci. Advances 9, eadd8856.

PubPeer Weblink: https://pubpeer.com/publications/C22E0805CB0B55CB7388F488611145

#1 (March 2024)

Accusation: One set of representative images in Figure 4B shows enlarged views of two synapses that cannot be found next to each other in the low-magnification view shown in the paper. The accusation is that these images represent image manipulations. In order to illustrate the accusation, the accuser drew new boxes into the published figure that are not in the paper.

Resolution: As explained in the figure legend, these are representative synapse images taken from the same experiment but are not adjacent in the low-magnification images shown. The low-magnification image is shown specifically to illustrate adjacent synapses whereas the high-magnification images is shown to illustrate particular features of synapses. All raw data were submitted to a public database (https://purl.stanford.edu/nb252dn4150). We showed on PubPeer now the entire dSTORM low magnification image that contains both illustrative synapses. PubPeer is establishing new fundamentalist rules whereby illustrative figures become the main point of papers instead of illustrations of experiments for readers.

Classification: Unfounded

#4, #6 & #7

Accusation: These accusations make the same point as #1: The commenters allege that it is unethical that we did not explicitly state in the figure legend that the representative images shown were not adjacent to each other in the section.

Resolution: Journal restrictions on legend sizes make it impossible to explain every detail but there was clearly no intent of hiding the fact that the representative images are just that, representative images, that were selected from a larger set of images. Otherwise we would have indicated this using customary boxes.

Classification: Unfounded

#10 & #12

Accusation: The commenters criticize that the leak currents in the experiments for Figure 3 is too high and allege that they cannot reproduce the graph in Figure 3B from the raw data that we made publicly available.

Resolution: Leak currents are always high when a high Cl^- concentration is used in the internal solution during recordings – this is normal. One of the co-authors who did not perform the original experiments for Figure 3B reanalyzed the raw data for Figure 3B and arrived at the same conclusion as that stated in the paper, finding it difficult to understand why the anonymous accuser could not reach that conclusion.

Classification: Unfounded

***

17. Paper: Wang, S., DeLeon, C., Sun, W., Quake, S.R., Roth, B.L., and Südhof, T.C. (2024) Alternative Splicing of Latrophilin-3 Controls Synapse Formation. Nature 626, 128-135.

PubPeer Weblink: https://pubpeer.com/publications/A04E94FAF81B5D7EC9E6B1668085EA

#1a (February 2024)

Accusation: The p values in Figure 5 must be wrong because they are the same for Exon 31 (E31) and Exon 32 (E32) conditions.

Resolution: The P values in Fig5 (and also throughout the paper) are identical for E31 and E32 in the same comparison groups because the splicing of E31 and E32 are mutually exclusive. Therefore, each PSI datapoint in E31 plot always has a corresponding datapoint with the value of 100-PSI in E32 plot, and for the same comparison group (e.g. KCl 0hr vs 6hr), the p value for E31 must be equal to E32.

Classification: Unfounded

#1b  (February 2024)

Accusation: The p values shown in Figure 5 are different from those of the Supplemental Tables and therefor one of them must be wrong.

Resolution: Figure 5 used a t-test as specified in the legend. The supplemental tables use Tukey’s test to calculate pairwise p values after correcting family-wise error as specified again in the legend. In the “Statistics and reproducibility” section we explicitly stated: “Most statistical tests were performed using two-sided t-tests, as indicated. To control for family-wise error during multiple comparisons, two-sided Tukey’s tests were used in parallel and the adjusted P values are summarized in Supplementary Tables 1 and 2, and do not change the conclusions drawn from t-tests in this work.”

Classification: Unfounded

#1c  (February 2024)

Accusation: Figures 5E and 5F are duplications because they are the same graph rotated 180 degrees.

Resolution: The distribution of Figure 5e and 5f are expected to be precise mirror-images of each other because E31 and E32 are mutually exclusive.

Classification: Unfounded

#3  (April 2024)

Accusation: We reported mis-labeling of a Figure in the paper committed by Nature staff as an illustration that published papers also contain errors introduced during processing

Resolution: No need for corrections except if Nature is convinced by PubPeer tweets that it should correct its own mistake

Classification: Unfounded

***

16. Paper: Woerman AL, Stöhr J, Aoyagi A, Rampersaud R, Krejciova Z, Watts JC, Ohyama T, Patel S, Widjaja K, Oehler A, Sanders DW, Diamond MI, Seeley WW, Middleton LT, Gentleman SM, Mordes DA, Südhof TC, Giles K, Prusiner SB. (2015) Propagation of Prions Causing Synucleinopathies in Cultured Cells. Proc. Natl. Acad. Sci. USA 112, E4949-4958.

PubPeer Weblink: https://pubpeer.com/publications/F80D8161AB29FEF18967FAF9A0D228#2

#1, 3-5 (December 2023)

Accusation: Based on data reconstructions, the statistical significance of panel A of Figure 6 is p = 0.0679 instead of p<0.05 as stated in the paper. Again, the accusation is supported by an echo chamber of commentators.

Resolution: The incriminated data are not from the Südhof lab but small differences in reconstructed data points could easily be responsible for the tiny difference in calculated p values, especially since figures are generally constructed from data points by programs and subsequent shuffling of panels introduces inaccuracies in figures. Given that the alleged p value difference is small, the conclusion that the stated p value is wrong seems to be unjustified, although this argument was criticized by PubPeer as inappropriate.

Classification: Unfounded

***

15. Paper: Jiang, X., Sando, R., and Südhof, T.C. (2021) Multiple signaling pathways are essential for synapse formation induced by synaptic adhesion molecule. Proc. Natl. Acad. Sci. U.S.A. 118, e2000173118. PMCID: PMC7826368

PubPeer Weblink: https://pubpeer.com/publications/027E93962D3C5DB86482283739C67D

#1 (November 2023)

Accusation: The stated statistical significance of the right graph in Figure 5C must be wrong because the error bars overlap

Resolution: Prism software indicates that the two conditions are significantly different, consistent with the fact that overlap of confidence intervals is not a reliable indicator of statistical significance (Schenker, Nathaniel, and Jane F. Gentleman. 2001. “On Judging the Significance of Differences by Examining the Overlap Between Confidence Intervals.” The American Statistician 55 (3): 182–86. http://www.jstor.org/stable/2685796.)

Classification: Unfounded

#3-6 (November 2023)

Accusation: Using the error bars as a guide to reconstruct the statistical significance between the two conditions, the two conditions cannot be significantly different

Resolution: We went back to the original data and confirmed that the error bars are SDs, not SEMs, and that the two conditions are statistically significantly different. An error in the figure legend was identified.

Classification: Minor error in figure legend

#8, 9, & 11 (March 2024)

Accusation: The same control was used in two different papers

Resolution: Correct – the experiments were carried out at the same time with the same controls

Classification: Unfounded

***

14. Paper: Ho, A., Morishita, W., Hammer, R.E., Malenka, R.C., and Südhof, T.C. (2003) A role for Mints in transmitter release: Mint 1 knockout mice exhibit impaired GABAergic synaptic transmission. Proc. Natl. Acad. Sci. U.S.A. 100, 1409-1414.

PubPeer Weblink: https://pubpeer.com/publications/866AA1811F89014742E1EAEB2BD25D

#1 (October 2023)

Accusation: The 60 day data point in Figure 3C cannot be statistically significantly different because of the error bars almost overlap.

Resolution: In the incriminated graph only the day 60 day data point is statistically significantly different. This is not a biologically significant result as discussed in the paper, but the existing statistical significance was nevertheless reported as mandated by publishing rules. Also see #1 under Paper 15 above.

Classification: Unfounded

#2 (October 2023)

Accusation: The fact that neurexin protein levels are lower in heterozygous than in homozygous KO mice in Table 1 is suspicious.

Resolution: Protein measurements are notoriously noisy. As a result, small changes in protein levels are not interpretable, especially if they were statistically not significantly different as in this case.

Classification: Unfounded

***

13. Paper: Biederer, T., Cao, X., Südhof, T.C., and Liu, X. (2002) Regulation of APP-dependent transcription complexes by Mints/X11s: Differential functions of Mint isoforms. J. Neurosci. 22, 7340-7351.

PubPeer Weblink: https://pubpeer.com/publications/34D6A8F36DFC9F19D753DCAF6B96FE    

#1 (October 2023)

Accusation: The lack of error bars on panel D of Figure 3 raises concerns about the validity of the data

Resolution: Error bars are not visible in Figure 3D because the error bars are too small

Classification: Unfounded

***

12. Paper: Fernandez-Chacon, R., Shin, O.-H., Königstorfer, A., Matos, M.F., Meyer, A.C., Garcia, J., Gerber, S.H., Rizo, J., Südhof, T.C., and Rosenmund, C. (2002) Structure/function analysis of Ca²⁺-binding to the C₂A-domain of synaptotagmin 1. J. Neurosci. 22, 8438-8446.

Weblink: https://pubpeer.com/publications/1FD9572D003130580F6C598C39D9C6

Date of accusation: September 2023

Accusation: Unknown since comment was removed by ‘moderator’

Resolution: No criticism recorded

Classification: Unfounded

***

11. Paper: Trotter, J.H., Hao, J., Maxeiner, S., Tsetsenis, T., Liu, Z., Zhuang, X., and Südhof ,T.C. (2019) Synaptic Neurexin-1 Assembles into Dynamically Regulated Active Zone Nanoclusters. J. Cell Biology 218, 2677-2698. PMCID: PMC6683742

PubPeer Weblink: https://pubpeer.com/publications/71F24BE796880C3A39AC29501382AB

#1 (July 2023)

Accusation: Control blots in Figure 7C are duplicated

Resolution: Original blots document that the control blots were likely not duplicated even though the blots show similar blotting artifacts as documented, although given the similarity of the blots one might easily conclude that they might be identical. A common error of PubPeer posts is that blots with similar artifacts are alleged to be duplicated, which neglects the fact that samples analyzed on the same blotting apparatus with the same antibodies exhibit similar artifacts even though they are different.

Classification: Unfounded

Postscript: Upon reviewing the primary data, we discovered a blot mixup in the images shown that we corrected with an Erratum but that was not detected on PubPeer

Classification: Minor error identified by the lab

#4 (October 2023)

Accusation: One bar in the Figure 9C graph is not labeled as significantly different but looks like it should have been labeled as significantly different

Resolution: PRISM software analysis of the data suggests that it is not significantly different

Classification: Unfounded

#6 (March 2024)

Accusation: Dr. Bik acknowledges that we published a correction not of any mistake PubPeer identified, but of a mistake we identified

Resolution: No resolution needed – the comment was just meant to amplify the fact that we found and acknowledged a mistake

Classification: Unfounded

#8 (March 2024)

Accusation: Dr. Bik alleges that there is a mistake in Figure S6E because two representative images overlap. As often, her comments are then echoed and amplified by Dr. Bazargan and Actinopolyspora

Resolution: The overlap exists but is perfectly legitimate since the panels illustrate sample images from the same experiment

Classification: Unfounded

***

10. Paper: Burré, J., Sharma, M., Tsetsenis, T., Buchman, V., Etherton, M., and Südhof, T.C. (2010) a-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro. Science 329, 1664-1668.

PubPeer Weblink: https://pubpeer.com/publications/2452F555579F6D021205B875814D82

#1 (May 2023)

Accusation: The blots in Figure 2B, 4B, and 4C are duplicated

Resolution: Original blots demonstrate that the blots are not duplicated. This is the same common error in PubPeer accusations as in paper 11, which alleges blot duplications based on similar artifacts but neglects the fact that similar samples run on the same blotting apparatuses exhibit similar artifacts

Classification: Unfounded

#3 (May 2023)

Accusation: Demands higher resolution images of the blots that we presented in PubPeer

Resolution: We feel the original blots we presented on PubPeer are sufficient to demonstrate that the accusation that the blots were duplicated is mistaken.

Classification: Unfounded

#5 & #6  (March 2024)

Accusations: Dr. Bik reinforced/repeated #1 and #3 accusations.

Resolution: The first author of the paper now posted the original blots of the paper to document that there is no evidence that the control blots were duplicated and that these blots exist.

Classification: Unfounded

#9  (January 2025)

Accusations: Dr. Sholto David alleges that the incriminated blots were manipulated based on his image analysis that identified microduplications

Resolution: Above we show a complete comparison of the published data, alleged manipulation, and high-resolution images of the submitted data which clearly shows that the allegation is wrong. This case is a great example to illustrate the nature of many false PubPeer accusations: In older papers, less sophisticated software led to changes in images that consist of duplications and other aberrations which are clearly not intentional and are not present in the original data. The problem we face as scientists is that for many old papers, original data are simply not available, which is not due to our fault. Given that social media like PubPeer seem to imply we are guilty if we can’t prove otherwise, this creates a toxic atmosphere especially for young scientists.

Classification: Unfounded

***

9. Paper: Patzke, C., Brockmann, M.M., Dai, J., Gan, K.J., Grauel, M.K., Fenske, P., Liu, Y., Acuna, C., Rosenmund, C., and Südhof, T.C. (2019) Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses. Cell 179, 498-513. PMCID: PMC7159982

PubPeer Weblink: https://pubpeer.com/publications/4AEAAAE084C8DFE9E26107D350B0B5

#1, 2, 4 & 5 (March 2023)

Accusation: The source data files for Figures 6E, 6F, 6K, S6F, S6G, S6Q, S6R, and S7C contain instances of data duplication.

Resolution: The accusation is largely correct. During assembly of the source data files, copy-paste errors of several blocks of numbers were committed for which a Correction has been filed. In addition, isolated number identities occurred that are not errors but intrinsic to the scientific method used.

Classification: Minor mistake identified by PubPeer

***

8. Paper: Lin, P.Y., Chen, L.Y., Zhou, P., Lee, S.H., Trotter, J.H., and Südhof, T.C. (2023) Neurexin-2 restricts synapse numbers and restrains the presynaptic release probability by an alternative splicing-dependent mechanism. PNAS 120, e2300363120

PubPeer Weblink: https://pubpeer.com/publications/DAF32F6DB6C166337E5381F769AE52

Note: This paper elicited a large number of comments because, as we confirmed in our reanalysis of the raw data deposited here (https://purl.stanford.edu/cp231wr9194), some of the source data were not properly analyzed, copy-paste errors were committed during assembly of the data, and the drafting of some figures was incorrect because the copy-paste errors were transmitted into the figures. Moreover, as always many comments were censored by the ‘Moderator’ who usually disallows author comments but generally does not censor accuser comments. Finally, a key author of the study was medically challenged during data assembly and the intense time of PubPeer criticism and provided incorrect answers on PubPeer.  

#1 - #4, #7, #11, and #13-#16 (March & April 2023)

Accusation: The source data files for Figures 2-6 contain extensive data duplications

Resolution: The accusation is correct. The data duplications initially appeared to be a minor mistake due to extensive copy-paste errors, but reanalysis of the data (see below) showed that the errors were transmitted into the figures. Moreover, the paper’s 1^st author then posted a ‘corrected’ set of replacement data that were not previously seen by the lab and did not appear to be from the Südhof lab, suggesting they were not based on a scientific analysis. This was the trigger that led to a reanalysis of the raw data by the lab. The reanalysis showed that the data duplications in the source data files were copy-paste errors made during assembly of the summary file resulting from an original analysis of the raw data that was not correctly performed, and that the copy-paste errors were erroneously included in the figures.

Classification: Data duplications are a minor error, but the original raw data analysis and figures include major errors that were not detected by PubPeer comments but uncovered in our re-analysis – PubPeer only identified the copy-paste errors and the implausible nature of the replacement data posted by the 1^st author.

#18 - #21, #23-#26, #28, #30, #31, #33-#35, #37, #38, #40, #42, #44, and #46 (April-August 2023)

Accusation: The unpublished replacement data posted by the 1^st author contain irregularities suggesting possible falsification

Resolution: The accusation is correct. The replacement data were not reviewed or endorsed by the Südhof lab prior to being posted by the 1^st author and their origin is unclear.

Classification: Post does not refer to a Südhof lab publication

#64-#67 and #69 (October 2023)

Accusation: The posted raw data may or may not contain technical issues

Resolution: The discussion here was largely based on an incomplete understanding of electrophysiological methods as one of the comments points out. Electrophysiological results are inherently noisy.

Classification: Unfounded

#68 (October 2023)

Accusation: The review of the paper was flawed because both reviewers are alumni of the Südhof lab

Resolution: The accusation is incorrect since Prof. Josh Huang was never associated with, or collaborated with, the Südhof lab, although Prof. Katsuhiko Tabuchi was in the Südhof lab 20 years ago but has since established a successful large independent research program as a full professor in Japan.

Classification: Unfounded

Postscriptum: The Südhof lab posted as response #70 (October 2023) the following statement:

“My lab has surveyed the many criticisms of this paper and reanalyzed part of the raw data. We observed multiple errors. The ones we know of, both discovered by us and raised by others, I’m listing here. Our preliminary conclusions are:

a. The published source data and the raw data associated with the paper are often inconsistent.

b. There are extensive raw data falling into two categories: raw data that were labeled as used for the paper and raw data that were not used from analogous experiments without a clear difference in quality or rationale.

c. The limited reanalysis of anonymized data confirms the conclusions of the paper, although we have analyzed only a small part of the data and the conclusions are not always statistically significant for all parameters. The absolute values of the mean parameters differ as would be expected when raw data are analyzed independently. Needless to say that does not confirm the paper as we also need to relate the data to the original lab notebook.

d. As regards comment #68, although Prof. Katsuhiko Tabuchi was a fellow in my lab 25 years ago, there has been no connection to my lab since then, and Prof. Josh Huang would indeed be surprised to learn that he was supposed to have been a fellow in my lab.

Overall, we concur that there are major inconsistencies with the source data of this paper compared to the existing raw data, which we are puzzled by. Note that the raw data, unlike blots or images, cannot be manipulated and contain identifiable metadata. Our current preliminary conclusion thus is that, consistent with some of the comments, the source data of this paper contain major flaws that cannot be explained by copy-paste mistakes as I had mistakenly surmised earlier but must have another origin.”

An ’Editorial Expression of Concern’ was published by PNAS that was immediately re-published as comment #71 on PubPeer and in accusatory blogs on ForBetterScience and Spectrum websites.

#72 and #73 (October 2023)

Accusation: The mean value in Figure 1B does not fit the datapoints associated with it, suggesting that the mean value is wrong

Resolution: The accusation is correct that the mean control value does not fit the datapoints but incorrect in concluding that the mean value is wrong. Replotting of the raw data in the source file clearly shows that the datapoints were accidentally shifted during construction of the figure, probably because in the adobe illustrator software independent image objects are often linked and movement of an object can inadvertently cause movement of other linked objects.

Classification: Minor mistake identified by PubPeer

Update. We retracted the P.Y. Lin et al. paper after our independent analysis of the raw data in 2024 (https://www.pnas.org/doi/10.1073/pnas.2403021121) with the following statement: “We wish to retract the paper because re-analysis of the original raw data for Figs. 2, 4, and 6 (https://purl.stanford.edu/cp231wr9194) revealed that, although our analyses of the original data are supportive of the conclusions of the paper, unresolvable differences exist between these raw data and the published data source file that cannot be corrected by a simple erratum. In addition, the data source file contained copy-paste errors, and Fig. 1 included shifted data points that occurred during figure drafting. We thank Dr. Daniel Matus of Stanford University for his independent analysis of the primary raw data."

Further analyses revealed two mistakes associated with the publication of this paper, a partly incorrect analysis and the incorporation of copy-paste mistakes from data files into figures.

***

7. Paper: Dai, J., Liakath-Ali, K., Golf, S., and Südhof, T.C. (2022) Distinct Neurexin-Cerebellin Complexes Control AMPA- and NMDA-Receptor Responses in a Circuit-Dependent Manner. E-Life 11, e78649.

PubPeer Weblink: https://pubpeer.com/publications/68D8490A4754CE00F936214C3931F2

#1 & #4 (March 2023)

Accusation: The source data file of the paper contains duplicated values for several cells

Resolution: The accusation is correct. The duplications are copy-paste errors that were corrected with an Erratum in the journal.

Classification: Minor mistake identified by PubPeer

#5, #7 & #8 (November 2023 & July 2024)

Accusation: An anonymous commentator confirms an Erratum was published, another commentator demands all raw data, and finally Dr. Bik 9 months later re-confirms that an Erratum has been published.

Resolution: The confirmation is correct but we are puzzled why it was posted except if it was meant to amplify a questioning signal.

Classification: Irrelevant comment

***

6. Paper: Zhang, X., Lin, P.Y., Liakath-Ali, K, and Südhof, T.C. (2022) Teneurins Assemble into Presynaptic Nanoclusters that Promote Synapse Formation via Postsynaptic Non-Teneurin Ligands. Nature Comm. 13, 2297.

PubPeer Weblink: https://pubpeer.com/publications/EC9A138F8BFAAE1F2FB803106703AB

#1 (March 2023)

Accusation: Two columns in the source data file for Figure 8 are duplicated

Resolution: The accusation is correct. The columns were accidentally duplicated in a copy-paste error during assembly of the file for sharing after figures were drafted. The error was corrected with an erratum. All raw data were submitted to a public database (https://doi.org/10.25740/kq306bq2466)

Classification: Minor mistake identified by PubPeer

#5 & #6 (July 2024)

Accusation: Dr. Bik, followed as usual by Dr. Bazargan’s ‘animation’, finds that two of the 413 fluorescence images of the paper may exhibit a partial overlap.

Resolution: We could not confirm the alleged overlap despite the animation but the complete raw data are publicly available for reanalysis (see https://doi.org/10.25740/kq306bq2466)

Classification: Unfounded

***

5. Paper: Dai, J., Patzke, C., Liakath-Ali, K., Seigneur, E., and Südhof, T.C. (2021) GluD1, A signal transduction machine disguised as an ionotropic receptor. Nature 595, 261-265.

PubPeer Weblink: https://pubpeer.com/publications/0B87E141DC8DFFF4826A9250A94BAD

#1 (February 2023)

Accusation: The PPR data in the source file are missing

Resolution: The corresponding data were mislabeled as belonging to ‘1f’ instead of ‘1k’ in the source data file due to a typing error. This mistake is being addressed in a ‘Correction’ in the journal.

Classification: Unfounded

#2 & #4 (February & March 2023)

Accusation: The source file contains duplicated values for two rows of numbers

Resolution: Correct. We introduced two copy-paste mistakes during transfer of data from experimental logs to the source file, as occurs when the ‘copy’ key is not pressed sufficiently. This mistake is being addressed in a ‘Correction’ in the journal.

Classification: Minor mistake identified by PubPeer

#3, #9, & #11 (March & June 2023)

Accusation: Some of the full-sized blots in Suppl. Figure 1b and 1c do not correspond to the cropped or quantified data in the Extended Data figure.

Resolution: Correct. Reassessment of original blots identified two related mistakes. In Suppl. Figure 1b one blot was mislabeled and not all blots were included. In Suppl. Figure 1c a different blot of the same experiment with identical results was shown. Again, these mistakes are being addressed in a ‘Correction’ in the journal.

Classification: Minor mistake identified by PubPeer

#12-#16 & #18 (July & November 2023)

Accusation: The data on the effect of a mutation on a function should not have been tested by t-tests

Resolution: T-tests are the standard test for manipulations that contains only a single independent variable and only compare that variable to the control, but not among samples. The graph reports a comparison of multiple single tests to the same shared controls, and could be plotted as multiple graphs consisting of a control and test condition. We agree that the question of the appropriate statistical test can be contentious especially if one focuses less on the biological experiment and more on the graph format but feel that the t-test is appropriate here.

Classification: unfounded

***

4. Paper: Sclip A, and Südhof, T.C. (2020) LAR receptor phospho-tyrosine phosphatases regulate NMDA-receptor responses. E-Life 9, pii: e53406. PMCID: PMC6984820

PubPeer Weblink: https://pubpeer.com/publications/613AFEC1A22C0725BB6D5A9E5CFE76

#1 (February 2023) & #4 (April 2023)

Accusation: Alleged duplication of blots and falsification of data because (i) quantifications of blots were made on the basis of different ‘n’s’ for different proteins but (ii) only a single Tuji control blot is shown for the quantifications.

Resolution: Ad (i), the ‘n’s’ (number of replicates) differ between experiments because each protein quantification is performed separately with a different antibody using the same samples. Different numbers of repeat experiments are performed for various proteins because the noise levels differ between proteins, with the noise level in turn being determined by a protein’s abundance and the quality of an antibody. Each antibody is different, and each immunoblotting quantification is a separate experiment compared to the same controls run on the same gels for multiple antigens. Ad (ii), a single sample control blot is shown for each set of experiments because illustrating samples of the Tuji control blots separately for each protein seems superfluous and adds no information. The same control blot is shown for panels B and C because these experiments were run on the same gels to illustrate that fact.

Classification: Unfounded

#7 & #9 (July 2024)

Accusation: Drs. Bik and Bazargan (in yet another ‘animation’) restate a year later the accusation that the control blot in panels B and C was duplicated, even though we explained already above that they are purposely the same. Dr. Bik states “The lengthy explanations given above [our earlier response in PubPeer, when we still tried to discuss data with PubPeer accusers, which is clearly impossible] seem to not address the concern raised above in #1, which is that two Tuji blots representing different samples (presynaptic vs active zone) look unexpectedly similar. I understand that each protein-of-interest might have been normalized using its own Tuji re-probe, and that protein quantifications are difficult, but that still does not explain the abovementioned similarity. Can the authors please address the actual concern? Also, these figures are not "for illustration purposes only" - they are the data.”

Resolution: Although our explanation was lengthy, we apologize that apparently it wasn’t lengthy enough. First, the samples analyzed are NOT different, they are the same. Second, these samples are run on the same gels and analyzed on the same blots and thus share Tuji controls. The two Tuji blots are thus ‘expectedly similar’ because they are identical. Third, we show the same Tuji blot for pre- and postsynaptic proteins because these proteins were analyzed on the same gels. This is real data but illustrative data – we do not show every blot we ever did. All this was described in earlier responses. We realize that Drs. Bik and Bazargan do not focus on the actual science and how science is done, and we appreciate the many questions they raise. We also realize that in order to not attract Drs. Bik’s and Bazargan’s accusations, we should choose different Tuji examples even though they are run on the same gels. At this point, scientists in our labs are becoming paranoid that something may look ‘unexpectedly similar’ and cause PubPeer comments that can destroy careers without having any impact on the science.

Classification: Unfounded

#12 (July 2024)

Accusation: An anonymous commenter understood that the samples are not different, for which we are grateful

Resolution: We appreciate that another commenter reads our responses

Classification: helpful comment

***

3. Paper: Dai, J., Aoto, J., and Südhof, T.C. (2019) Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA- and AMPA-Receptor Responses. Neuron 102, 993-1008. PMCID: PMC6554035

PubPeer Weblink: https://pubpeer.com/publications/568B4CF8B40A979424B7F343F3B061

#1 (February 2023)

Accusation: The y-axis labels of Figure 6B and 6C are incorrect

Resolution: Correct - the y-axes were mislabeled

Classification: Minor mistake identified by PubPeer

#3-#11, #22, #23  (February & March 2023)

Accusation: The source data for Figure 2D, 4A, 5E, 6A, 6B, 8, S2, S4, and S8 contain duplications and possible calculation errors

Resolution: Correct - the source data contain multiple isolated errors produced by copy-paste mistakes that also lead to errors in downstream calculations but have no discernable effect on figures or conclusions.

Classification: Minor mistake identified by PubPeer

#25-#28 and #30   (March 2023)

Accusation: The statistical tests used for experiments examining 3 experimental groups – derived from littermate SS4+ and SS4- mice vs. unrelated WT samples – are generally incorrect

Resolution: The accusers express their view that the three experimental groups should be treated as equivalent in pairwise comparisons whereas biologically they are not equivalent. The 3 experimental groups comprise a genetically distinct WT sample expressing both SS4+ and SS4- variants of neurexins and two genetically identical samples that express either only SS4+ or SS4- variants of neurexins. Thus, the statistics are more complex than a simple 2-way ANOVA with a post-hoc correction since the comparison of the two genetically identical samples between each other is inherently different from their comparison with the WT sample. Given this experimental configuration, we believe our statistical approach may be the most appropriate, but it is possible that a non-traditional test that accounts for the experimental non-equivalency might be even better.

Classification: Unfounded

#33-#37  (March 2024)

Accusation: Dr. Bik states that the Correction we published on this paper to rectify the labeling mistakes and copy-paste errors in our paper represent a ‘Mega Correction’ and another accuser criticized that we did not address the accuser’s concerns about statistics.

Resolution: We feel that our correction of copy-paste errors in supplementary tables and of mislabeled supplementary graphs is not a ‘Mega Correction’, although we regret these errors. The impact of our 'Mega Corrections' is purely procedural and has no consequences for the actual science.

Classification: Irrelevant comment

***

2. Paper: Sclip, A., Bacaj, T., Giam, L., and Südhof, T.C. (2016) Extended synaptotagmin (ESyt) triple knock-out mice are viable and fertile without obvious endoplasmic reticulum dysfunction. PlosONE 11, e0158295.

PubPeer Weblink: https://pubpeer.com/publications/FBC43D21E0E903A65AF81CD8D1CAF1

#1 (August 2022)

Accusation: Alleged duplication of blots in Figure 3 and S1.

Resolution: It is correct that the same tubulin and actin blots were used as loading controls for experiments that were performed at the same time. This is not a duplication of blots but the use of the same controls for an experiment that was performed at the same time, and we should have noted this in the legends. An Erratum to document this detail is being prepared.

Classification: Minor mistake identified by PubPeer

#3 (December 2022)

Accusation: A correction was made in the published paper without notification

Resolution: This is incorrect – no correction was made

Classification: Unfounded

#5 (December 2022)

Accusation: Claims that the data are no longer in the PlosONE website

Resolution: A screenshot from the PlosONE website shows that the data are clearly still there.

Classification: Unfounded

#7 (June 2023)

Accusation: The Munc18 and Syt1 blots are identical, i.e. represent duplications

Resolution: Original blots of the experiments are now shown on PubPeer demonstrate that the Munc18 and Syt1 blots were derived from the same experiments run on the same gels but probed with species-specific distinct antibodies. Since these proteins have similar sizes, the shape of their bands is nearly identical, creating an appearance of duplication. However, based on direct communication of the accusers with PlosONE ‘Ethics’, the PlosONE ‘Ethics’ administrators demand that we amend the blots to show samples from the same original publicly deposited blots that make the bands look different, which will be published as a ‘Correction’. The ‘Correction’ doesn’t actually correct anything but journals are very sensitive to PubPeer criticism and their Ethics departments are tasked with eliciting Corrections.

Classification: Unfounded

#10 (August 2023)

Accusation: Maintains that he/she/they cannot see the blots properly

Resolution: Original blots were publicly deposited (https://purl.stanford.edu/vq040hz0549)

Classification: Unfounded

#12 (March 2024)

Accusation: Dr. Bik states “the blot provided in #2 appears to contain munc18 bands in green and syt1 bands in red, just under the munc18 bands, while the published panels show both bands separately (no double bands) and both in red. So it appears you either did not provide the originals, or the published panels have been color-converted with double bands removed.”

Resolution: We have already published a ‘Correction’ that explains better the various blots with inclusion of full-length original blots and boxed areas, and all original blots have been posted on PubPeer and are also publicly available at the published URL as high-resolution images. However, the accusations that "So it appears you either did not provide the originals, or the published panels have been color-converted with double bands removed" are incorrect. In the incriminated blot, two different species-specific antibody signals were monitored on the same blot (for Munc18 and synaptotagmin that have almost identical molecular weights) in different optical channels. Thus, no double band has been removed, nor are colors converted because every color represents an arbitrary assignment of a digital signal. We chose to show the Munc18 and synaptotagmin blots in the same false color in our paper because we thought that this would make it easier to compare the blots, but this has clearly confused people who are not familiar with this type of experiments.

Classification: Unfounded

#13 (July 2024)

Accusation: Dr. Bik quotes the Correction

Resolution: No resolution necessary since this comment is just meant to amplify the number of comments on PubPeer.

Classification: Unfounded

***

1. Paper: Yi, F., Danko, T., Botelho SC, Patzke C, Pak C, Wernig, M., and Südhof, T.C. (2016) Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons. Science 352, aaf2669.

PubPeer Weblink: https://pubpeer.com/publications/CBAA10B0FBF31CC41E9B7D56C8B0C3

#1 (April 2018)

Accusation: Since the resting membrane potential of the human neurons derived from stem cells in the experiments is at approximately -40 mV, the neurons must be leaky and sick and it is therefore not possible to draw any conclusions from these neurons

Resolution: All human neurons produced from stem cells are immature and exhibit a decreased resting potential, even though they are not leaky or sick. They exhibit robust active and passive membrane properties and form fully functional synapses

Classification: Unfounded

#3 (April 2018)

Accusation: How can different clones in different sets of experiments produce very similar readouts given that evoked EPSC amplitudes are dependent on intensity of stimulation

Resolution: The electrophysiological approaches used here were validated previously in a number of papers and shown to be highly reproducible across experiments (e.g., see Zhang, Y., Pak, C.H., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Xu, W., Yang, N., Patzke, C., Chen, L., Wernig, M., and Südhof, T.C. (2013) Rapid Single-Step Induction of Functional Neurons from Human Pluripotent Stem Cells. Neuron 78, 785-798. PMCID: PMC3751803; Pak, C., Danko, T., Mirabella, V.R., Wang, J., Liu, Y., Vangipuram, M., Grieder, S., Zhang, X., Ward, T., Huang, Y.W.A., Jin, K., Dexheimer, P., Bardes, E., Mittelpunkt, A., Ma, J., McMachlan, M., Moore, J.C., Qu, P., Purmann, C., Dage, J.L., Swanson, B.J., Urban A.E., Aronow, B.J., Pang, Z.P., Levinson, D.F., Wernig, M., and Südhof, T.C. (2021) Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-Derived NRXN1-Mutant Neurons. Proc. Natl. Acad. Sci. U.S.A. 118, e2025598118. PMCID: PMC8179243)

Classification: Unfounded

#4 (April 2018)

Accusation: The electrophysiological experiments are unreliable (e.g., “Anyone knows that hyperpolarizing MP from -40 to -70 takes heroic current magnitude” and “Not all cells should be active at rest, even not ~60 %”)

Resolution: The electrophysiological approaches used here are consistent with a large number of previous studies by multiple independent experimenters not only the Südhof lab but also in others, and the data shown in the paper are internally fully consistent

Classification: Unfounded

#5 (April 2018)

Accusation: Mean amplitudes and intervals do not correlate with the 'representative traces'

Resolution: Electrophysiological measurements vary between cells and representative traces are only an illustration of the results, they do not depict an average of traces

Classification: Unfounded

#10 & #11 (July 2024)

Accusation: Desires to know the current used to measure the resting membrane potential

Resolution: We unfortunately do not have the resources to research all questions posed by the audience if there is no pressing rationale

Classification: Unfounded