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.

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ACADEMIC EDITOR:

While the manuscript presents a valuable methodological contribution, both of the reviewers have suggested that a revision is needed to improve the visualization and enhance the practical impact of the work. Please revise the manuscript accordingly and incorporate the reviewers' suggestions.

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Please submit your revised manuscript by May 07 2025 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Mahdi Roozbeh

Academic Editor

PLOS ONE

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3. Thank you for stating the following financial disclosure:

This study was supported by the Health Development Promotion Project-Anesthesia and Critical Care Research Project (KM-20231120-01), Guangdong Medical Research Fund Project (A2024728、A2024723), the Zhanjiang Science and Technology Research Project in 2022 (No: 2022A01197), and the Science and Technology Development Special Fund Competitive Allocation Project of Zhanjiang City (No: 2021A05086).

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: N/A

Reviewer #2: Yes

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: The author reported an interesting study that utilized transcriptomic datasets to identify disulfidptosis-related genes involved in PAH. Below are my comments and suggestion for the manuscript:

1. For a better understanding of the study and datasets, I suggest providing a clear description and discussion of the nature of the datasets used.

2. The findings presented in Figure 1 require further elaboration.

3. Further elaboration is needed for each panel in Figure 2. What do the modules represent? Do they correspond to genes? How many genes are included in each panel? Why is the grey module considered insignificant?

4. Figure 3, Is there a correlation between the outcomes of GO enrichment analysis and KEGG enrichment analysis?

5. What criteria led to the selection of the green module over the yellow module for further analysis? Panel G, the list does not seem too extensive. Please provide the gene names instead of numbers.

6. Figure 5, please state the differences between (panel A & panel B) and (panel C & panel D).

7. The sentence on page 9/29, starting with “Figure 6 showed that the high...” until the end, is unclear. Please revise it for clarity.

8. Figure 7, the Fiddle diagram: What is the meaning of “Con” and “Treat”? What kind of differences exist between the cells? Are they related to cell number, gene expression, or other factors?

9. The discussion is lengthy and lacks a well-organized flow. I suggest focusing more on explaining the findings and linking them to the disease.

Reviewer #2: In this work, Chen and colleagues utilized GEO datasets to investigate the expression of disulfidptosis-related genes in arterial hypertension (PAH). After constructing gene modules with WGCNA, they performed GO and KEGG analyses of the module with increased significance to disease pathology. They applied LASSO, Random Forest, and SVM-RFE in parallel to narrow down the list of hub genes to a couple of overlapping genes (USP32 and ZNF655). They further used GSEA to determine potential pathways and mechanism functions of the hub genes, performed immune infiltration analysis to determine immune cell distributions between control and pulmonary hypertension groups, and built ceRNA regulatory network for USP32 and ZNF655 to predict related miRNA and lncRNA regulatory networks.

Major concerns:

The figures with higher resolution (text is not completely visible in figures 2, 6, 7, and 8, for example), and more detailed legends are necessary (including experiment descriptions, and how the graphs were generated). This is essential to fully visualize and understand the data, as well as follow the interpretation of the results.

The authors present interesting data produced through a well structured and logical analysis, yet limited by sample size and lack of experimental validation. USP32 and ZNF655 are involved in multiple cellular processes and allow for generating many hypotheses about their role in PAH, therefore, the relevance of their findings could be strengthen by focusing the discussion primarily on how the data connects with what is already well known about the cell and molecular biology of PAH (instead of tackling multiple connections not necessarily as robust).

Minor concerns:

In “Dynamic module identification isolated three significant modules—green, yellow, and gray—with the gray module deemed nonsignificant. Hierarchical clustering further identified the green module as particularly relevant to disease

pathology”, can you explain why gray module was deemed nonsignificant? And why is green module is relevant to disease pathology?

In Figure 2, groups are divided into ‘control’ and ‘treat’. Can you explain what “treat” means?

In Figure 4 legend, reference to C is missing.

In Figure 5, what is the difference between A and B, and C and D?

Duplicated sentences in discussion: “Furthermore, studies involving bovine subjects have revealed that the expression of olfactory receptors (ORs) is governed by MOR4, a member of the GPCR superfamily. MOR4 is primarily recognized as a binding site for the zinc finger (ZNF) transcription factor gene family, suggesting a link between GPCR activity and ZNF[41]. Furthermore, studies involving bovine subjects have revealed that the expression of olfactory receptors (ORs) is governed by MOR4, a member of the GPCR superfamily. MOR4 is primarily recognized as a binding site for the zinc finger (ZNF) transcription factor gene family, suggesting a link between GPCR activity and ZNF [42].”

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

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 submit your revised manuscript by Jul 23 2025 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.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

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,

Mahdi Roozbeh

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: All reviewer comments have been adequately addressed, and I am satisfied with the author's responses.

Reviewer #2: The authors have made efforts to address previous comments; however, I still recommend that they clarify the following key points to improve the clarity of their findings:

1. Please clarify whether p-values reported for enrichment analyses, correlation studies, and other statistical tests were adjusted for multiple comparisons (e.g., using FDR or Bonferroni correction).

2. While the construction of ceRNA networks for USP32 and ZNF655 is a potentially valuable addition, the analysis lacks prior introduction or justification for the relevance of miRNA/lncRNA in pulmonary hypertension. Please add a brief explanation and provide the methodological details for this analysis.

3. As noted previously, the discussion is overly broad and lacks a cohesive focus. It attempts to associate USP32 and ZNF655 with numerous biological pathways and mechanisms, many of which are tangential or speculative, and dilute the impact of their findings. Consider to streamline the discussion to emphasize a few well-supported, relevant pathways that directly connect their findings to pulmonary hypertension. This would be especially important given that there is no experimental validation of the involvement of USP32 or ZNF655 in pulmonary hypertension in this paper.

Other comments:

Figure 4G would be more informative if it contained the gene names instead of numbers.

Figures 6, 7a and 7c remain difficult to read. Please consider improving its resolution.

“Zinc lipoprotein (ZNF) family” appears to be a misstatement. Please revise for accuracy.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

Comprehensive analysis of disulfidptosis-related genes in pulmonary hypertension through machine learning and immune infiltration: spotlight on USP32 and ZNF655 as key regulators

PONE-D-25-07068R2

Dear Dr. Dingyu 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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mahdi Roozbeh

Academic Editor

PLOS ONE

Comments to the Author

Reviewer #2: All comments have been addressed

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

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

Reviewer #2: The authors' response addresses all previous concerns and suggestions - particularly, the discussion is now much more focused and objective.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: No

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