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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 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: 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: The research article “JMJD3 regulates the M2 macrophage polarization and promotes the growth of breast cancer cells via STAT6/IRF4 axis” by Juan Lyu et al investigates the role of JMJD3 in promoting M2 macrophage polarization through the STAT6/IRF4 signaling axis and draws a correlation between breast cancer progression and M2 TAMs.

Using data from over 1,000 breast cancer patients from the Cancer Genome Atlas (TCGA), the authors found that high levels of M2 macrophages correlate with advanced clinical features (e.g., age >60, ER+/PR+ status, late-stage tumors) and poorer overall survival.

In vitro, the authors induced M2 macrophage-like polarization by exposing THP-1 monocytes to PMA, IL-4, and IL-13. These M2 macrophage-like cells showed increased expression of JMJD3, IRF4, and phosphorylated STAT6. Knockdown of JMJD3 or inhibition of STAT6 reduced the expression of IRF4 and M2 markers while overexpression of JMJD3 enhanced M2 polarization and further supported cancer cell growth.

Mechanistically, the study demonstrates that JMJD3 promotes M2 polarization through the STAT6/IRF4 pathway. STAT6 activation triggered by IL-4/IL-13 increases JMJD3 expression, which in turn enhances IRF4 transcription. Disruption of this axis using a STAT6 inhibitor AS1517499 or JMJD3 knockdown resulted in impaired M2 polarization. In turn, MDA-MB cancer cells exposed to conditioned media of polarized THP-1 cells whose STAT6/JMJD3/IRF4 pathway had been disrupted showed decreased proliferation and increased apoptosis.

MINOR REVISIONS

THP-1 is monocytic cell line that can be differentiated into macrophage-like cells. The authors should refrain from classifying differentiated cells as macrophages and instead classify them as macrophage-like. Point in case, macrophages do not typically produce IL-2. Therefore, the inclusion of IL-2 measurement in figure 2D and referring to IL-2 as an M2 marker is incorrect and should be revised. Typically, M2 macrophages are characterized by IL-10, IL-4, and IL-13 secretion.

Line 199/200: please provide a reference for the statement.

How do the authors account for the practically neglectable differences in apoptosis of MDA-MB-231 cells when subject conditioned medium from polarized THP-1/macrophages overexpressing or lacking JMJD3?

The underlying concept of the paper is the role of JMJD3 in catalyzing the demethylation of H3K27me3 and promoting gene transcription. However, at no point in their model of silencing or overexpressing JMJD3 the authors actually show the effects on H3K27me3 demethylation. The authors should consider doing so as it would further validate their observations and strengthen the paper.

In the proposed model (Figure 6) the authors include CD163. However, at no point in the paper CD163 is mentioned. As such it should be removed from the model. Furthermore, the inclusion of “Cytokine” is not helpful when the authors know the cytokines in question: IL4 and IL13. Please substitute cytokine by IL4 and IL13 and make sure to use different colors for the dots, so that it is easily understood that there are two cytokines involved. I also find that the blue arrows that indicate what processes are occurring are not very intuitive. The authors might want to consider improving that.

Finally, the authors need to provide data points for figures 2B, 2F, 3H, 3J, 3L, 4C, 4G, 5A, 5B, and 5C. Data points should also be more visible in all the figures. Additionally, the authors need to provide information on how many times the essays were done, if we are looking at biological repeats or technical repeats.

Reviewer #2: 1. TCGA analysis showed higher M2 scores in ER-positive and PR-positive patients. However, the functional assay was performed on the MDA-MB-231 cell line (ER-negative, PR-negative). Would the authors could explain about this? Does the STAT6/JMJD3/IRF4 signaling axis have a different or more prominent role in different breast cancer subtypes?

2. The authors should supplement with other cell lines, for example using conditioned media on breast cancer cell lines representing different subtypes such as MCF-7 (ER-positive, PR-positive) or SKBR3 (HER2-positive) to test whether the growth-promoting effect depends on the breast cancer subtype.

3. The study used the pharmacological inhibitor AS1517499 to inactivate STAT6. Small molecule inhibitors can sometimes have off-target effects. To increase the robustness of the results, did the authors consider confirming the results using genetic methods, such as using shRNA or siRNA to knockdown STAT6?

4. The signaling pathway is presented in a linear direction: STAT6 → JMJD3 → IRF4. However, do the authors think it is possible that IRF4 or one of its target genes could negatively regulate the activity of STAT6 or JMJD3?

5. The study demonstrated that JMJD3 knockdown reduced M2 markers. However, the spectrum of macrophage differentiation is considered to be a continuum rather than a discrete M1/M2 state. The question is: does JMJD3 inhibition simply block the M2 differentiation pathway, or does it actively push macrophages towards M1 polarization?

6. The authors could examine M1-specific markers (e.g., iNOS, IL-12) in JMJD3 knockdown cells, as this would provide further insight into the role of JMJD3 as a “switch” that directs polarization.

7. JMJD3 is a histone demethylase that can act on many different genes, not just IRF4. The study focused on the JMJD3/IRF4 axis. So do the authors think that JMJD3 can regulate other genes involved in M2 macrophage function in parallel with regulating IRF4?

8. The authors suggest that STAT6 inhibition reduces JMJD3 expression, suggesting that STAT6 regulates transcription of the JMJD3 gene. However, this is only an indirect relationship. Experiments are needed to demonstrate whether STAT6 acts on the promoter region of the JMJD3 gene to activate transcription (Chromatin Immunoprecipitation - ChIP).

9. The study only used conditioned medium to demonstrate that M2 macrophages affect cancer cells through the soluble factors they secrete. However, direct physical interactions between the two cell types may also play an important role. Direct co-culture of macrophages with breast cancer cells could be used to assess whether the observed effects are enhanced.

10. The authors have not specified which factors secreted by M2 are primarily responsible for promoting cancer cell growth.

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Reviewer #1: Yes: Marco CraveiroMarco Craveiro

Reviewer #2: No

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Submitted filename: PONE-D-24-45718 - Review suggestions.docx

JMJD3 regulates the M2-like macrophage polarization and promotes the growth of breast cancer cells via STAT6/IRF4 axis

PONE-D-24-45718R1

Dear Dr. Zhang,

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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Li Yang, M.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Thanks for the authors' efforts to comprehensively improve your manuscript according to editor's and reviewers' comments. I am pleased to inform you that your paper can be accepted for publication now.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: The authors have been very proactive and professional in addressing concerns and suggestions raised in the previous review. The revised manuscript and the authors' detailed "Reviewer Response" document have addressed nearly all of the Reviewers' comments.

The authors added new experiments that confirm the demethylase activity of JMJD3 by demonstrating its effect on H3K27me3 levels, which strongly validates the proposed mechanism.

The addition of functional data using the MCF-7 (ER-positive) breast cancer cell line resolves previous discrepancies with the TCGA findings and reinforces that the STAT6/JMJD3/IRF4 axis plays a tumor-preserving, tumor-supporting role across different breast cancer subtypes.

The authors have also revised the model (Figure 6) and ensured that all figures now include individual data points and clarified that all experiments were performed on at least three independent biological replicates, improving transparency and reproducibility.

The authors openly acknowledge the remaining limitations (such as the lack of direct ChIP evidence of STAT6-JMJD3 binding and the need for in vivo validation) and are committed to exploring these questions through future experiments.

I have no further comments.

Reviewer #3: You may consider a systems biology approach or choose to use computational methods in future. I HiC-seq would give a genome-wide assessment of chromatin modification which would be helpful in your case.

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Reviewer #2: No

Reviewer #3: No

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