Dear Dr. Alizadeh,

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Academic Editor

PLOS One

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“This work was part of the first author's Ph.D. dissertation, completed under the supervision of Effat Alizadeh and Masoud Darabi. This study was supported by the Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 66664) and the University Medical Center Schleswig-Holstein, Lübeck, Germany.”

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Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was part of the first author's Ph.D. dissertation, completed under the supervision of Effat Alizadeh and Masoud Darabi. This study was supported by the Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 66664) and the University Medical Center Schleswig-Holstein, Lübeck, Germany.”

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“This work was part of the first author's Ph.D. dissertation, completed under the supervision of Effat Alizadeh and Masoud Darabi. This study was supported by the Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 66664) and the University Medical Center Schleswig-Holstein, Lübeck, Germany.”

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“This work was part of the first author's Ph.D. dissertation, completed under the supervision of Effat Alizadeh and Masoud Darabi. This study was supported by the Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 66664) and the University Medical Center Schleswig-Holstein, Lübeck, Germany.”

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: No

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Reviewer #1: The study explores a highly relevant topic in metabolic and regenerative medicine: the paracrine signaling of brown and white adipocytes via exosomes. The use of four different human cell models (OGCs, MCF-7, hADSCs, hUCMSCs) provides a comprehensive overview of how these secretomes influence diverse biological processes, from cancer apoptosis to fertility and stemness. The integration of lipidomic profiling adds significant depth to the phenotypic observations.

The characterization of isolated hADSCs and their subsequent differentiation into BALCs and WALCs is well-documented using flow cytometry, qPCR, and Western Blotting.

The study doesn't limit itself to phenotypic changes but seeks a metabolic explanation through mass spectrometry-based lipidomics. The differential effects (e.g., BALCs-Exo increasing estradiol in OGCs while CM reduces it) are striking and suggest complex regulatory mechanisms.

While the paper establishes what happens, the how remains largely correlative. Identifying specific exosomal cargo (miRNAs or proteins) responsible for the observed effects would significantly strengthen the impact. Moreover, for vesicle characterization, endoplasmic markers such as calreticulin and/or calnexin should be added to exclude contamination of vesicles derived from the endoplasmic reticulum. In figure 7A, change “Annexin 3” with “Annexin V”. Then, the discussion should be more concise, prioritizing the interpretation of key findings and expanding the speculation regarding the potential molecular/proteic content of the vesicles.

Reviewer #2: This manuscript investigates the effects of conditioned media (CM) and exosomes derived from brown adipocyte-like cells (BALCs) and white adipocyte-like cells (WALCs) on different human cell models, including ovarian granulosa cells (OGCs), MCF-7 breast cancer cells, hADSCs, and hUCMSCs. The topic is relevant and potentially interesting because adipocyte-derived extracellular vesicles are increasingly recognized as mediators of intercellular communication. The inclusion of lipidomics also increases the translational value of the study.

However, although the manuscript contains potentially valuable observations, the current version remains descriptive and preliminary.

In particular, the exosome characterization, mechanistic validation, and biological rigor require substantial improvement before the manuscript can be considered suitable for publication.

The authors identify the isolated particles as “exosomes”, but the characterization is incomplete according to current extracellular vesicle (EV) guidelines (MISEV recommendations). Only morphology (TEM/SEM), size, zeta potential, and positive markers (CD9, CD63, TSG101) are shown.The evidence is not sufficient to exclude contamination from proteins, apoptotic bodies, lipoproteins, or other EV populations, especially because a precipitation kit was used for isolation.The authors should add:

- Nanoparticle tracking analysis (NTA) to determine particle concentration and size distribution.

- Negative EV markers (e.g., calnexin, GM130, or albumin) to demonstrate purity.

- Quantification of exosome yield and normalization strategy (particle number or protein amount).

The manuscript assumes that target cells respond directly to adipocyte-derived exosomes, but there is no evidence that exosomes are internalized by recipient cells. The authors must perform exosome uptake assay using fluorescent labeling (e.g., PKH67, DiI, or membrane dye) and microscopy or flow cytometry.

The lipidomics section is interesting but highly speculative. Many conclusions regarding mitochondrial remodeling, oxidative phosphorylation, membrane specialization, or metabolic programming are not experimentally validated. To support these claims, authors should include functional metabolic assays, such as:

- Mitochondrial respiration analysis (Seahorse assay),

- ATP production assay,

- ROS measurements,

- Mitochondrial membrane potential assay.

The osteogenic differentiation conclusion relies mainly on ALP activity. However, ALP is an early marker and does not fully demonstrate osteogenic commitment. The authors should add Alizarin Red quantification and evaluation of osteogenic genes (RUNX2, Osteocalcin, Osteopontin).

English should be revised by a native speaker.

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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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Brown and white adipocyte like cells derived exosomes differentially regulate stemness, hormone secretion, apoptosis, and lipid metabolism in model cells

PONE-D-26-16792R1

Dear Dr. Alizadeh,

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,

Mohammad H. Ghazimoradi

Academic Editor

PLOS One

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: (No Response)

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

Reviewer #1: Yes

Reviewer #2: (No Response)

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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: (No Response)

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

Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

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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