-->PONE-D-25-21042-->-->EP300 promotes bladder cancer cell migration through SNAI2-->-->PLOS ONE

Dear Dr. Guo,

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This work was supported by the National Natural Science Foundation of China No.32070818 to C.M.G., the Xingdian talent support program of Yunnan Province to C.M.G. and Guangdong Hybribio Biotech Funding No.H20230314, H20230311 and H20230313 to C.M.G.

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Zhang Q. He C. and Guo C. are supported from National Natural Science Foundation of China No.32070818, Guangdong Hybribio Biotech Funding H20230314, H20230311 and H20230313.

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This manuscript investigates the mechanism by which EP300 promotes bladder cancer cell migration through SNAI2. The study design is reasonable, the experimental data are relatively sufficient, and the conclusions have certain scientific significance. After review, it is recommended to "Accept after minor revisions". The specific revision suggestions are as follows:

Regarding the supplementation of experimental details:

In the cell culture section, the source background information of the four bladder cancer cell lines (SW780, T24, RT4, and 5637) should be clarified, including their pathological grades, metastatic potentials, etc., to enhance the rationality of the selection of experimental models.

In the scratch wound healing assay, it is necessary to supplement the specific composition of the "serum-free complete medium" and the reason for choosing this condition for the experiment; in the Transwell assay, the reason for using the same concentration of A485 in both the upper and lower chambers should be clarified.

Regarding the improvement of result analysis:

It is mentioned in Figure 1 that "ac-EP300 is significantly increased in 2/4 tumor tissues". Specific statistical analysis results, including sample size and P-value, should be supplemented to more strongly support the association between EP300 activation and the malignant progression of bladder cancer.

In the RNAseq analysis, for the 229 commonly downregulated genes, their specific functional classifications and direct evidence of association with SNAI2-related pathways should be supplemented, such as whether there are target genes of SNAI2.

Regarding the deepening of the discussion section:

The discussion mentions that EP300 may have oncogenic or tumor-suppressive effects in different cancers. Combined with the results of this study, it is necessary to further elaborate on the specificity and potential risks of targeting EP300 as a therapeutic strategy in bladder cancer.

The basis for selecting the concentration of A485 (10μM) in this study should be supplemented, such as whether it has been verified by pre-experiments, as well as references regarding the possible safety and effectiveness of this concentration in vivo.

Regarding data availability and ethical statements:

The data availability section mentions that all data are fully available without restriction. Specific access methods, such as database names and accession numbers, should be supplemented.

In the ethical approval section, it is necessary to clarify whether informed consent was obtained from the participants during the collection of human samples and the form of consent (written or oral).

Please revise and improve the manuscript according to the above comments. If the responses are appropriate, this manuscript is suitable for publication in PLOS ONE.

Reviewer #2: This study investigates the role of EP300 in bladder cancer migration. It confirms the positive association between EP300 and bladder malignancy. The study observed A485, which is a small-molecule inhibitor of EP300, significantly inhibited the migration of multiple bladder cancer cell lines (SW780, T24 ,RT4, 5637) both in scratch wound healing and Transwell assays, and disrupted organoid migration in 3D culture. Using bulk RNA-seq analysis and western blotting the research found that A485 downregulates SNAI2-related signaling pathways and reduce the expression of SNAI2 and the H3K27ac level. Overexpression of SNAI2 enhances the bladder cancer cell migration, and EP300 can promote bladder cancer cell migration via SNAI2, and targeting EP300 could be a potential therapeutic strategy.

1、Lack of in vivo validation for the effects of EP300 inhibition. Effects the translational medical value of the conclusion.

2、The results only used 4 matched pairs of bladder cancer and adjacent tissues. Is the sample size too small?

3、The discussion says that the EP300 has two opposite effects, but the study only explores the EP300’s role of promoting migratory, and don’t delve into the possibility of EP300 suppressing the bladder cancer, nor does it analyze the influence of these opposite effects for EP300-targeted therapeutic strategies.

4、The discussion only repeats the results of the study, lacks an analysis of the limitation of the study.

5、Figure5, only inhibition of EP300 via A485 alone led to a decrease in SNAI2 expression was observed. Lack the direct evidence to demonstrate that EP300 binds to the promoter or enhancer region of SNAI2. It is recommended to supplement the study with experiments(such as ChIP-qPCR and luciferase reporter assay) to validate the regulatory relationship.

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Reviewer #2: No

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-->PONE-D-25-21042R1-->-->EP300 promotes bladder cancer cell migration through SNAI2-->-->PLOS One

Dear Dr. Guo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.-->

• Please revise your work as per the reviewers' comments.

-->Please submit your revised manuscript by Jan 30 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

-->If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Chien-Feng Li, M.D., Ph.D.

Academic Editor

PLOS One

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: No

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Recommendations to Strengthen the Evidence for EP300-SNAI2 Mediated Migration in Bladder Cancer

The manuscript by Zhang et al. investigates the role of EP300 in promoting bladder cancer cell migration via the transcription factor SNAI2. The study employs a multi-faceted approach, including IHC on patient samples, in vitro 2D/3D migration assays, in vivo mouse models, RNA-seq, and molecular biology techniques. The central finding—that the EP300 inhibitor A485 suppresses migration by downregulating SNAI2—is novel and of potential interest to the field of bladder cancer therapeutics. However, the manuscript in its current form requires revisions to strengthen the mechanistic claims, improve data rigor, and enhance clarity before it can be considered for publication.

Major Concerns:

1. Insufficient Mechanistic Evidence

How can you demonstrate that EP300 directly regulates SNAI2, rather than through indirect pathways? The use of inhibitors and H3K27ac CUT&Tag alone is insufficient to prove direct regulation. Can you provide EP300-specific ChIP experimental data? Furthermore, can SNAI2 overexpression fully reverse the inhibitory effect of A485 on migration, thereby proving it is the key downstream effector of EP300?

2. Over-reliance on a Single Experimental Approach

The entire study heavily depends on a single inhibitor, A485. To exclude potential off-target effects, can you provide genetic evidence (e.g., EP300 knockdown or knockout) demonstrating that the observed phenotypes (inhibited migration and downregulated SNAI2) are consistent with pharmacological inhibition?

3. Inadequate Support for Conclusions

The manuscript states that A485 only shows a "trend" in vivo without statistical significance. Under these circumstances, why is this still presented as key supporting evidence? Please provide detailed sample sizes (n values) and statistical methods, and discuss the impact of this non-significant result on your conclusions.

4. Lack of Essential Control Experiments

How have you verified that the inhibition of migration by A485 is not simply due to cytotoxicity or proliferation suppression? Please provide cell viability/proliferation data under the exact same conditions used in the migration assays as supplementary information.

5. Data Presentation Issues

Please comprehensively check and correct all figure citations throughout the manuscript.

Reviewer #2: Your study elegantly identifies the EP300-SNAI2 axis in bladder cancer cell migration, with solid in vitro, 3D culture, and in vivo evidence. The revised data, including expanded clinical samples (12 pairs) and GSA-deposited RNA-seq/CUT&Tag data (PRJCA038992), significantly strengthens the work. However, two key issues require attention. First, per PLOS ONE’s mandates, uncropped, unadjusted blot/gel images (e.g., Western blot for SNAI2 and H3K27ac) remain unpublished—please deposit these in the supplementary materials or a public repository. Second, the in vivo BBN model shows a trend of reduced invasion but lacks statistical significance, possibly due to small sample size (n=4 per group); expanding the cohort or adding supplementary functional assays (e.g., metastasis tracking) would reinforce translational relevance.

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Reviewer #1: No

Reviewer #2: No

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EP300 promotes bladder cancer cell migration through SNAI2

PONE-D-25-21042R2

Dear Dr. Chunming Guo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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