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PLOS One

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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: Yes

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

Reviewer #1: Yes

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: No

Reviewer #2: Yes

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4. 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: In this paper, we reveal the role of ROS-related gene features in ischemic stroke through systems biology methods, and propose CLEC4E as a potential immune regulator, which is innovative. However, there are some problems to be solved.

1. Supplementary method details

Specify the sample size of the cohort, quality control (PCA analysis), data preprocessing steps (such as batch effect correction, whether the data is preprocessed), and WGCNA module definition threshold (such as dynamic shear tree height).

2. The WGCNA in Figure 2 is an analysis using the GSE58294 or GSE16561 cohort. Please describe it clearly.

Which cohort was used for the LASSO regression analysis in Figure 4?

Please describe which cohort was used for the immune infiltration analysis in Figure 7?

3. The study fails to thoroughly explore how CLEC4E connects ROS with immune infiltration (e.g., receptor-ligand interactions and downstream signaling pathways). It is necessary to supplement the comparison with existing biomarkers (e.g., NSE and S100B).

4. The potential applications of gene signatures in early diagnosis, prognostic stratification, or therapeutic target development were not discussed. A comparison with existing biomarkers (e.g., NSE and S100B) should be supplemented.

5. Human Data Ethics Statement : The document fails to specify whether the GEO data has undergone ethical review. A supplementary statement regarding the ethical compliance of data sources should be included.

6. Completeness of supplementary materials : While uncropped Western blot images are labeled as "not for publication", critical experimental data should be made publicly available to ensure reproducibility. It is recommended that these key data be fully presented in the supplementary materials.

7. Please provide the code for all analysis.

Reviewer #2: This manuscript integrates two independent transcriptomic cohorts to map reactive oxygen species (ROS)–related programs in ischemic stroke and to derive a compact gene signature with diagnostic/predictive utility, complemented by immune-deconvolution analyses and in vivo validation.

1. Please provide a more explicit description of each GEO cohort (sample sizes, tissue/source, timing relative to stroke onset, key clinical covariates, and inclusion/exclusion criteria), as these factors can materially influence transcriptomic patterns and immune estimates. Also clarify how potential batch effects across platforms/cohorts were handled (or why not needed), how missing clinical information was operationalized, and whether any confounder adjustment was performed beyond quality control.

2. The analysis is anchored to five curated ROS-related gene sets; however, readers will benefit from a clear rationale for choosing these specific sets and how overlapping genes across sets were handled. Consider adding sensitivity analyses using alternative ROS resources (or broader oxidative-stress annotations) and report whether the key module and the final gene signature remain stable under reasonable variations in ROS gene set definitions.

3. Because WGCNA results can vary with parameter settings, please report key choices (soft-threshold selection criteria, minimum module size, merge thresholds) and provide more explicit module preservation outputs. It would also help to clarify whether the network was constructed on all genes vs. ROS-filtered genes (and the trade-offs), and to demonstrate that the “brown module” association is not driven by cohort-specific artifacts.

4. The manuscript would be strengthened by a clearer statement of the prediction target (diagnosis vs. risk vs. prognosis) and a consistent modeling description. Ensure that feature selection, model fitting, and hyperparameter tuning are fully nested within cross-validation to avoid optimistic bias. In addition to AUC, please report clinically relevant metrics (e.g., sensitivity/specificity at prespecified thresholds, PPV/NPV with plausible prevalence, and calibration diagnostics) and justify how decision-curve analysis was parameterized for this setting.

5. Deconvolution methods can diverge depending on signatures and tissue context. Please quantify concordance across algorithms, discuss assumptions (especially if derived from blood expression profiles), and consider additional checks (e.g., whether observed correlations persist after controlling for global inflammation markers). The negative association between CLEC4E and B/T cell infiltration is intriguing, but causal language should be avoided without mechanistic experiments.

6. It would be useful to briefly situate your pathway/functional enrichment pipeline within the landscape of contemporary large-cohort, reproducible enrichment workflows by adding a few representative citations (e.g., https://doi.org/10.1038/s41467-025-61785-z; https://doi.org/10.1186/s12916-023-02920-9; https://doi.org/10.1503/jpn.220202). This small addition would help readers benchmark your enrichment choices (gene-set resources, multiple-testing control, and interpretation strategy) against established population-scale practice.

7. In the Statistical analyses section, consider referencing several large-sample database studies that use closely related modeling and validation strategies (e.g., https://doi.org/10.1016/j.dcn.2025.101566; https://doi.org/10.1016/j.psychres.2025.116503; https://doi.org/10.1192/bjp.2023.107).

8. Please provide additional detail on randomization/blinding, sample sizes and power considerations, timepoints, and whether endpoints were prespecified. To better connect the computational findings to mechanism, consider functional perturbation (e.g., knockdown/overexpression or pharmacologic modulation) targeting CLEC4E (or its upstream/downstream signaling) and report whether this alters ROS burden, immune infiltration markers, and neuronal injury outcomes in the model.

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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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Attachments

Attachment

Submitted filename: Reviewer s reply.pdf

Systems Biology Analysis Uncovers a ROS-Associated Gene Signature and Immunomodulatory Role of CLEC4E in Ischemic Stroke

PONE-D-25-51535R1

Dear Dr. Ding,

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,

Yuzhen Xu

Academic Editor

PLOS One

Additional Editor Comments (optional):

I am pleased to inform you that your manuscript has been accepted following peer review.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: Yes

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Reviewer #1: Great job! The author has thoroughly addressed all my questions and provided many solutions. I have no further questions.

Reviewer #2: The authors have satisfactorily addressed all of my previous comments and concerns. I am pleased with the revisions and recommend this manuscript for publication.

Reviewer #3: (No Response)

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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

Reviewer #3: No

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