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PLOS ONE

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Additional Editor Comments (if provided):

The manuscript "Exosomes from bone marrow mesenchymal stem cells protect melanocytes under vitiligo-related conditions through induction of NRF2/HO1 expression" is interesting; however, as reviewers stated, it needs to be revised according to their suggestions. Therefore, it is advisable to revise the manuscript in light of the reviewers' comments.

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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: 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: Yes

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: No

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Reviewer #1: The manuscript is of good understanding towards the vitiligo and has potential to be carried in geenrating the therapeutic applications. Its well written and informed.

For improvement, manuscript may also provide details of the MSCs characterization as no detail on surface markers is given. Further, details about the role of each agent used in the vitiligo study and its therapeutic effect with the exosomes may be given.

Reviewer #2: Title: Exosomes from bone marrow mesenchymal stem cells protect melanocytes under vitiligo-related condition through induction of NRF2/HO1 expression

This is a well-designed and timely study that investigates a promising cell-free therapeutic strategy for vitiligo. The research question is clear and clinically relevant. The manuscript provides compelling in vitro evidence that exosomes derived from Bone Marrow Mesenchymal Stem Cells (BMSCs-Exos) can protect melanocytes from oxidative stress—a key driver of vitiligo pathogenesis—by activating the NRF2/HO1 antioxidant pathway. The experimental design is logical, and the data generally support the conclusions. The findings could have significant implications for developing novel treatments for vitiligo.

Strengths

1. The study addresses a significant unmet need in vitiligo treatment by exploring BMSCs-Exos as a safer, "off-the-shelf" alternative to whole MSC transplantation, mitigating risks like low engraftment survival and potential tumorigenicity.

2.The authors thoroughly evaluated the protective effects of BMSCs-Exos by assessing multiple endpoints: cell viability, apoptosis, and a panel of oxidative stress markers (GSH/GSSG, SOD, ROS, MDA). This multi-faceted approach strengthens the conclusions.

3. The use of conditioned medium from BMSCs treated with the exosome inhibitor GW4869 (BMSCs-GW4869) is a critical and well-considered control. It helps to attribute the observed effects specifically to the exosomes themselves rather than other soluble factors secreted by BMSCs.

4. The use of siRNA to knockdown *NRF2* expression is the strongest part of the manuscript. It effectively demonstrates that the protective effects of BMSCs-Exos are dependent on the NRF2/HO1 pathway, providing causal evidence for the proposed mechanism.

Shortcomings

1.The authors note that BMSCs-GW4869 still had a significant effect on apoptosis (Fig 2C), which they correctly attribute to potentially incomplete inhibition of exosome release. This is a limitation. The manuscript would be strengthened by including data (e.g., NTA or Western blot of markers) showing the actual reduction in exosome yield in the GW4869-conditioned medium to quantify the efficacy of inhibition.

2. The entire study is conducted in an in vitro cell line model (PIG3V). While this is a valid starting point, the conclusions about therapeutic potential for vitiligo would be significantly more powerful if supported by data from an in vivo animal model of vitiligo

3.The study identifies the NRF2/HO1 pathway as the mechanism but does not investigate what component of the BMSCs-Exos is responsible for activating this pathway. Is it specific miRNAs, proteins, lipids, or mRNAs? Identifying the active cargo would be a major advance and is a natural next step.

4.The findings are based on a single immortalized human melanocyte cell line (PIG3V). Repeating key experiments in primary human melanocytes would enhance the translational relevance and generalizability of the results.

5. The exosome inhibitor is consistently misspelled. It is GW4869, not "GW4849" (e.g., Abstract, Methods, Results, Figure legends).

6. Page 23, Line 3: "BMCs-Exos" should be "BMSCs-Exos".

7. Page 24, Line 1: "Bcl-X" is likely a typo and should be "BAX".

8. Page 25, Line 3: "NO-1" is a typo and should be "HO1".

9.The term "BMSCs-GW4849" is used in the text, but the figures (e.g., Fig 2, 3, 4) use the correct "BMSCs-GW4869". This must be harmonized.

10.The figure legends are very long and seemingly complicated, making them difficult to follow. The descriptions of each experimental group could be summarized in a table format within the legend or in a supplementary table for clarity.

11.The statistical annotation in Figure 5 is particularly complex and hard to decipher (e.g., `[5]`, `[55]`, `[&]`, `&&&`). Using more standard notations (e.g., asterisks and hashtags with clear definitions) would greatly improve readability.

12.In the Supplementary Figure legend (Page 35), it says "Fig 1B", "Fig 1C", etc. It should be "Fig S1B", "Fig S1C" for consistency.

13.While generally understandable, the manuscript would benefit from thorough proofreading by a native English speaker or a professional editing service to improve fluency. Some examples:

14.Page 11: "...causing white skin patches and reduced quality of life" -> "...causing depigmented patches on the skin and a reduced quality of life".

15.Page 12: "MSCs exhibit capacities of extensive proliferation..." -> "MSCs have a high capacity for extensive proliferation..."

16.Page 13: "Exosomes are safe without risk of tumorigenicity..." -> "Exosomes are safe, with no risk of tumorigenicity..."

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what does this mean?

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Exosomes from bone marrow mesenchymal stem cells protect melanocytes under vitiligo-related conditions through induction of NRF2/HO1 expression

PONE-D-25-38005R1

Dear Dr. Xu,

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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Kind regards,

Vikash Chandra, PhD

Academic Editor

PLOS ONE

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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: The manuscript is of importance as it focuses on an important aspect of the biology, vitiligo.

May be accepted as I feel it is ok

Reviewer #2: The authors' revisions are comprehensive and have successfully addressed all concerns raised by both reviewers. The manuscript is significantly improved in terms of scientific rigor, clarity, and presentation. This work makes a valuable contribution to the field by establishing a novel protective role and mechanism for BMSCs-Exos in vitiligo, providing a solid foundation for future research.

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