PONE-D-23-14523Lactate-induced histone lactylation by p300 promotes osteoblast differentiationPLOS ONE

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Reviewers' comments:

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Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #3: Yes

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

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors investigated the function and mechanism of histone lactylation on osteoblast differentiation. The results showed a role of histone lactylation in promotion of undifferentiated cells to osteoblast differentiation. The paper shows evidence that supports the conclusions, however, the following aspects should be addressed.

Major Concerns:

1. Figure1d and Figure 2d: the author only repeats once on KLA, it’s not enough in standard scientific article.

2. The author designed a LDH inhibitor (oxamate) group to study the function of LDHA on osteoblast differentiation, why not add a gene overexpression group?

3. There is no significant in results.

4. Figure 2 and 3a: Add relative intracellular lactate results.

Minor Concerns:

Figure 1b: Add scale in your picture. Check it through the MS.

Figure1d and Figure 2d: The KD of proteins should be added.

Reviewer #2: In this study, the authors demonstrated that lactate produced by glycolysis regulates osteoblast differentiation via histone H3 lactylation by promoting histone lactyl transferase activity by HAT/p300, which epigenetically enhances the expression of genes promoting osteoblast differentiation.

This is an interesting study, but some issues need to be clarified.

1. In Figures 1d through 4d, there are two bands stained with anti-lactyl lysine antibody (PanKla) in H3. What do these two bands represent and density ratio of which band was measured? In addition, there are no error bars for PanKla/H3 ratio. Because these are very important data to show H3 lactylation by lactate, these experiments need to be repeated and statistical significance should be presented.

2. In Figure 2, an LDHA inhibitor, oxamate, dose-dependently reduced extracellular lactate concentration and ALP activity, and 10 mM oxamate reduced lactyl lysine in H3 to a similar extent to that under low glucose (Figure 1d). However, Runx2 expression was inhibited only mildly and insignificantly, which is also the case in Figure 4c. Without change in Runx2 expression, how osteoblast differentiation was suppressed by oxamate?

3. In Figure 3, addition of lactate to low glucose medium recovered extracellular lactate concentration, ALP activity, PanKla/H3 ratio and Runx2 expression. In contrast, there was almost no change in Sp7 and Tnap expression. How ALP activity was increased despite very little change in Tnap expression, and what was the explanation for these discrepant results in Runx2, Sp7 and Tnap between Figures2 and 3? In Figures 2a and 3a, only extracellular lactate concentration was demonstrated. However, what is important is intracellular lactate concentration, and the authors should demonstrate intracellular lactate concentration.

4. In Figure 4, gene expression profiles of Runx2, Sp7 and Tnap were similar to those under oxamate treatment in Figure 2, and were different from those in Figure 3. These discrepant results under different treatments give readers a concern about the validity of the authors’ conclusion. In addition, knockdown of p300/HAT causes inhibition of histone acetylation and may change expression of various genes aside from change in histone lactylation.

5. It is worthwhile to refer to and discuss about the paper by Karsenty group (Wei J, et al. Cell 151: 1576-91, 2015) demonstrating that glucose uptake via Runx2-dependent expression of Glut1 is required and that Glut1 and Runx2 crosstalk determines osteoblast differentiation.

Reviewer #3: The study by Minami et al. describes the role of p300 lactylation as a mechanism associated with osteoblast differentiation. The authors conducted an experimental design using C2C12 cells to demonstrate the involvement of lactate in BMP-2-induced differentiation. While the experiments support this mechanism, there are some aspects that should be evaluated by the authors.

Considering that histone lactylation is key to understanding the regulatory mechanism in this article, it is noteworthy that only one result is shown in the bar graph of Figures 1, 2, 3, and 4 (d). It is suggested to include more replicates and display their dispersion as standard deviation (SD) along with statistical analysis. Were both bands considered for relative quantification since two bands are observed? Whatever the case may be, it is recommended to clarify this in the Materials and Methods section.

To demonstrate the role of LDHA and the conversion of pyruvate to lactate, oxamate was used. Was a cell viability analysis performed to rule out possible cytotoxic effects?

For the statistical analyses, the Student's t-test was used. However, in Figures 2 and 3, more than two experimental groups were used. In such cases, it is more appropriate to use analysis of variance (ANOVA) and post-hoc multiple comparisons tests. Additionally, significant differences are indicated in Figure 3C (Tnap/Gapdh) between low/0 and low/20 mM lactate, but the graph should be modified on its axis to visualize this unclear difference. Furthermore, in this figure, differences between high and low are shown for the Sp7/Gapdh gene, but no expression was detected in one group. It should be clearly stated which groups are being compared in this figure.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Lactate-induced histone lactylation by p300 promotes osteoblast differentiation

PONE-D-23-14523R1

Dear Dr. Sasa,

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,

Atsushi Asakura, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

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?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

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 #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?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: My comments were adequately addressed by the authors. The quality of the manuscript was improved after revision.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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: Yes: Xiangzhen Shen

Reviewer #2: No

Reviewer #3: No

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