Knockdown of KDM5B Leads to DNA Damage and Cell Cycle Arrest in Granulosa Cells via MTF1

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract: sentence from line 12 to 14 is difficult to follow until the end of the sentence, as the condition is at the end of the sentence (KDM5B knockdown). Please change the order in the sentence. Similar for line 21 to 24, it is difficult to follow.

 

Introduction: Deep changes must be considered in the introduction section. The authors have copied and pasted a section from the abstract of reference 9, from line 41 to line 46, which should be avoided. Furthermore, at line 41, the authors refer to cell proliferation, while at line 46-47, they refer to meiotic DSBs, but the connection between these concepts is not well explained.

 

Additionally, the literature cited at line 49-50 is insufficient for a paper on cell cycle arrest. Therefore, this paragraph should be revised to increase the number of literature citations on cell cycle regulation and arrest.

 

Line 52: Recently, xx reported... Please ensure that the text is thoroughly proofread by all authors before submission. There are additional examples of lack of attention to detail by the authors, such as the absence of a capital letter after a period at line 53, or a capital letter in the middle of a sentence at line 56, which does not make sense. 

 

Minor: Caenorhabditis elegans must be written in italics. C. elegans proteins must be cited properly (SPO-11) (both line 47)

 

 

Materials and Methods:

 

Minor:

Line 97. Capital letter.

Line 103: ^oC and Celsius.

Line 110. English (per)

Line 114. TransfEction.

Line 134. anti.

Table 3. Observed band…KDa?

 

Results:

 

Squares in Fig 1B, meaning?

 

Perform co-staining of KMD5B and marker FSHR to validate that KDM5B was localized in GCs.

 

Fig 1F. Quantification of “Relative fluorescence intensity”. Are the data relativized?

 

Same for Fig 1H.

 

Line 258. Explain why the next step is studying gene expression profiles. Results looks like a serial of bullet points without any relationship between them. This is an example but I have this feeling throughout the full text.

 

Instead of measuring BrdU incorporation by IF, it would be more informative to measure it with LSC. This would allow us to assess the rate of cell progression through the early and late sections of S-phase and identify the main problems after KDM5B knockdown, such as early triggering of origin of replication, replication elongation, and so on.

 

Line 309. γH2AX is a specific indicator for the detection of DNA damage. I recommend the authors could include relevant literature on this topic. The text mentions "expression of γH2AX", but it is actually the phosphorylation of H2AX that is being referred to.

 

Relative fluorescence of γH2AX is indicated, but it is important to note that any measurement is relative (Fig 4G). To be able to compare intensities, raw data should be used for quantification and pictures of different samples should be taken under the same conditions. The authors should clarify whether this was done in their study and clearly cite in the text.

 

In addition to measuring relative fluorescence, another common measure in the field is the quantification of foci. Therefore, I would suggest that the authors also quantify the number of γH2AX foci and include markers of DNA damage repair such as Rad51, RPA, and 53BP1 to strengthen the connection between increased DNA damage after KDM5B knockdown and levels of apoptosis.

 

Fig 7D. Using same reflection than before, is OE of MFT1 reducing the levels of DNA damage markers?

 

 

Some minor comments:

 

Line 255. Were not was

Line 248. Capital letter

Line 338. Please, provide more information to figure legends. Example. 4G doesn’t refers to the condition to increase gH2AX signal in the cell.

Line 334. The results indicated? that multimers indicated…(please, check the sentence)

 

 

Discussion:

The authors need to describe, analyze, and interpret their findings in relation to previous literature, but I don't think they have done so adequately. They have not explained the significance of their results and how they relate back to the research question(s). Instead of describing their findings one by one independently, the authors should join them together and give an overall idea of their results. Additionally, the authors should take care to avoid minor writing mistakes. Some sentences, such as sentences 437 to 439, are not acceptable.

Author Response

Abstract: sentence from line 12 to 14 is difficult to follow until the end of the sentence, as the condition is at the end of the sentence (KDM5B knockdown). Please change the order in the sentence. Similar for line 21 to 24, it is difficult to follow.

Thank you very much for your suggestion. We have modified the order of expression and moved the condition (KDM5B knockdown) to the first part of the sentence for easier understanding.

 

Introduction: Deep changes must be considered in the introduction section. The authors have copied and pasted a section from the abstract of reference 9, from line 41 to line 46, which should be avoided. Furthermore, at line 41, the authors refer to cell proliferation, while at line 46-47, they refer to meiotic DSBs, but the connection between these concepts is not well explained.

Thank you very much for your suggestion. We have modified the content of this part and summarized the literature cited. In addition, the relationship between meiotic DSBs and cell proliferation is elaborated in line 44-46.

 

Additionally, the literature cited at line 49-50 is insufficient for a paper on cell cycle arrest. Therefore, this paragraph should be revised to increase the number of literature citations on cell cycle regulation and arrest.

Thank you very much for your advice. We have added references to DNA DSBs related to cell cycle arrest in lines 53-55.

 

Line 52: Recently, xx reported... Please ensure that the text is thoroughly proofread by all authors before submission. There are additional examples of lack of attention to detail by the authors, such as the absence of a capital letter after a period at line 53, or a capital letter in the middle of a sentence at line 56, which does not make sense.

Thank you very much for your correction. We apologize for the mistake in the article. We have modified the above problems.

 

Minor: Caenorhabditis elegans must be written in italics. C. elegans proteins must be cited properly (SPO-11) (both line 47)

Thank you very much for your correction. We have modified these problems.

 

Materials and Methods:

 

Minor:

Line 97. Capital letter.

Line 103: ^oC and Celsius.

Line 110. English (per)

Line 114. TransfEction.

Line 134. anti.

Table 3. Observed band…KDa?

 

Thank you very much for your correction. We apologize for the oversight in the article. We have modified the above problems.

 

Results:

 

Squares in Fig 1B, meaning?

Thank you very much for your question. The squares in the image indicate granulosa cells, we have added this explanation to the figure legend.

 

Perform co-staining of KMD5B and marker FSHR to validate that KDM5B was localized in GCs.

Thank you very much for your suggestion. Please forgive us for not being able to do the co-staining experiment. Since both FSHR and KDM5B are rabbit antibodies, we cannot stain two colors of fluorescent secondary antibodies. Therefore, we first performed FSHR staining to identify granulosa cells, and then KDM5B staining. The cells used in the experiment were all granulosa cells collected in the same way, so we thought it would be convictive to stain them separately. We are sorry again that we cannot add this experiment. We hope our explanation can clear up your doubts.

 

Fig 1F. Quantification of “Relative fluorescence intensity”. Are the data relativized?

Same for Fig 1H.

Thank you very much for your question. Yes, the data are relativized. We used image J to obtain the fluorescence intensity of each cell and then analyzed the data. In this experiment, NC group and siKDM5B group were shot under exactly the same parameters and conditions, and we kept the original czi files for subsequent inspection.

 

Line 258. Explain why the next step is studying gene expression profiles. Results looks like a serial of bullet points without any relationship between them. This is an example but I have this feeling throughout the full text.

Thank you very much for your suggestion. After knocking down KDM5B, we detect phenotypes such as cell cycle and DNA damage, as well as RNA sequencing. The purpose is to explore which genes have changed in expression after knockdown of KDM5B, screen downstream target genes, and further explore the mechanism of action of KDM5B.

 

Instead of measuring BrdU incorporation by IF, it would be more informative to measure it with LSC. This would allow us to assess the rate of cell progression through the early and late sections of S-phase and identify the main problems after KDM5B knockdown, such as early triggering of origin of replication, replication elongation, and so on.

Thank you very much for your advice and we agree with you. LSC allows us to assess the rate of cell progression through the early and late sections of S-phase and identify the main problems after KDM5B knockdown. But please forgive us that due to limited experimental conditions and equipment, my laboratory cannot do LSC. So we are very sorry and we really appreciate that you can understand our situation.

 

Line 309. γH2AX is a specific indicator for the detection of DNA damage. I recommend the authors could include relevant literature on this topic. The text mentions "expression of γH2AX", but it is actually the phosphorylation of H2AX that is being referred to.

Thank you very much for your advice. We cited relevant literature on γH2AX in lines 315-316 and elucidated that γH2AX refers to phosphorylated H2AX. Thanks again for your advice!

 

Relative fluorescence of γH2AX is indicated, but it is important to note that any measurement is relative (Fig 4G). To be able to compare intensities, raw data should be used for quantification and pictures of different samples should be taken under the same conditions. The authors should clarify whether this was done in their study and clearly cite in the text.

Thank you very much for your advice. All fluorescence quantitative experiments in this experiment, NC group and siKDM5B group were shot under exactly the same parameters and conditions, and we kept the original czi files for subsequent inspection.

 

In addition to measuring relative fluorescence, another common measure in the field is the quantification of foci. Therefore, I would suggest that the authors also quantify the number of γH2AX foci and include markers of DNA damage repair such as Rad51, RPA, and 53BP1 to strengthen the connection between increased DNA damage after KDM5B knockdown and levels of apoptosis.

Thank you very much for your advice. We agree with you that there should be quantified statistics on γH2AX foci. Therefore, we modified the statistical method of Fig. 4G and 7E. We have quantified γH2AX foci, and then made comparison. At the same time, we also added changes in the expression of DNA damage repair marker genes, including RNA seq results and qRT-PCR results. We are very sorry that antibodies to these proteins of goat have not been found, and the sequence homology between mice with these genes and goat is not high. However, it takes too long and costs too much to customize goat antibodies, so WB and IF experiments cannot be supplemented. We are sorry again and we really appreciate that you can understand our situation.

 

Fig 7D. Using same reflection than before, is OE of MFT1 reducing the levels of DNA damage markers?

Thank you very much for your advice. We have quantified γH2AX foci, and then made comparison. OE of MFT1 reducing the levels of DNA damage markers.

 

Some minor comments:

 

Line 255. Were not was

Line 248. Capital letter

Thank you very much for your correction. We feel sorry for the mistake in the article. We have modified the above problems.

 

Line 338. Please, provide more information to figure legends. Example. 4G doesn’t refers to the condition to increase gH2AX signal in the cell.

Thank you very much for your advice. We have modified the expression of the figure legend of 4G.

 

Line 334. The results indicated? that multimers indicated…(please, check the sentence)

Thank you very much for your correction. We have modified multimers to JC-1 aggregates.

 

 

Discussion:

The authors need to describe, analyze, and interpret their findings in relation to previous literature, but I don't think they have done so adequately. They have not explained the significance of their results and how they relate back to the research question(s). Instead of describing their findings one by one independently, the authors should join them together and give an overall idea of their results. Additionally, the authors should take care to avoid minor writing mistakes. Some sentences, such as sentences 437 to 439, are not acceptable.

Thank you very much for your suggestion. We have revised the discussion section. We selectively reorganized several parts and added discussion of our experimental results. At the same time, in order to avoid similar problems, we checked the full text and corrected the errors. Thanks again for your advice!

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Yang et al, aim to elucidate the effect of KDM5B on goat granulosa cells by using siRNA, RNA-seq, and other methods. They found that KDM5B played an important role in the cell cycle and oxidative stress levels in goats granulosa cells. Importantly, they identified a new gene, MTF1, which may act as the target of KDM5B to regulate the cell cycle and DNA damage in goat granulosa cells. I believe the results is of interest. However, there are several suggestions need to be addressed before publication.

1. Since MTF1 was identified to play important roles in goat granulosa cells, it is highly recommended to add more information about MTF1 and its role in granulosa cells should be added.

2. In the Result section, description of the result part is a little monotonous, and some background introduction should be added and some experimental results should be explained.

3. As the authors performed RNA-seq to identify critical genes in goat granulosa cells, it is necessary to upload the original RNA-seq data to NCBI SRA or other platform.

4. The word ‘transfection’ spelled incorrectly on line 114. Kindly correct it.

5. The meaning of P < 0.01 was not explained in 2.13, but it was appeared in figure legend. Kindly add the specific meaning of P < 0.01.

6. The author mentioned that all experiments were repeated at least 3 times in 2.13, but I found that there were only two sets of repetitions for each treatment in Figure 2b. Could the authors further explain the discrepancy?

7. The significance of difference was not marked in figure 3E.

8. The position of the mark of BCL2 mRNA expression was not correct.

Author Response

In this manuscript, Yang et al, aim to elucidate the effect of KDM5B on goat granulosa cells by using siRNA, RNA-seq, and other methods. They found that KDM5B played an important role in the cell cycle and oxidative stress levels in goats granulosa cells. Importantly, they identified a new gene, MTF1, which may act as the target of KDM5B to regulate the cell cycle and DNA damage in goat granulosa cells. I believe the results is of interest. However, there are several suggestions need to be addressed before publication.

1. Since MTF1 was identified to play important roles in goat granulosa cells, it is highly recommended to add more information about MTF1 and its role in granulosa cells should be added.

Thank you very much for your advice. We have added an introduction about MTF1 in lines 81-87.

2. In the Result section, description of the result part is a little monotonous, and some background introduction should be added and some experimental results should be explained.

Thank you very much for your suggestion. We have revised the description of the result part and added some narration about the background.

3. As the authors performed RNA-seq to identify critical genes in goat granulosa cells, it is necessary to upload the original RNA-seq data to NCBI SRA or other platform.

Thank you very much for your advice. We have uploaded the original data of RNA seq to NCBI SRA.

4. The word ‘transfection’ spelled incorrectly on line 114. Kindly correct it.

Thank you very much for your correction. We apologize for the oversight in the article. We have modified the above problems.

 

5. The meaning of P < 0.01 was not explained in 2.13, but it was appeared in figure legend. Kindly add the specific meaning of P < 0.01.

Thank you very much for your suggestion. We have added the explanation of P < 0.01 in 2.13.

6. The author mentioned that all experiments were repeated at least 3 times in 2.13, but I found that there were only two sets of repetitions for each treatment in Figure 2b. Could the authors further explain the discrepancy?

Thank you very much for your suggestion. In RNA seq, we found a set of outliers. Therefore, we did not include this set in the analysis. We think the good repeatability between the two groups is convincing. Thank you again for your suggestion.

7. The significance of difference was not marked in figure 3E.

Thank you very much for your correction. We apologize for the oversight in the article. We have modified the above problems.

8. The position of the mark of BCL2 mRNA expression was not correct.

Thank you very much for your correction. We apologize for the mistake in the article. We have modified the above problems.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for your comments and the effort you have made. However, I have some additional concerns:

Lines 46-47: After a cell enters meiosis, it is not prone to DSBs, as there are programmed DSBs carried out by Spo11. Please clarify this sentence: Are there continuously occurring DSBs by SPO-11, or an accumulation of DNA breaks? Does this result in inhibited meiosis or failure of successful meiosis? Although there are mutations that lead to a massive accumulation of breaks, meiosis is still able to finish, although the oocyte may not be viable.

Lines 53-54: Reference 10 does not support this statement. Reference 10 discusses mouse meiosis. Please revise this sentence.

Line 54: Please (again) use Caenorhabditis elegans (in italics) and SPO-11 (not Spo-11).

Lines 57-58: Please revise this sentence.

Line 60: Please use "cell cycle progression" instead of "process".

Line 239: A P value < 0.01 indicates a more significant difference than a P value < 0.05.

Figure 4, page 13: Please provide Figure legends. In panel c, what do the black and grey bars represent?

A consideration for all the figures: In Fig 4C, 5B, and 5C, quantification is relative. For example, the protein levels in grey in Fig 4C are referred to as levels of the black ones, which are considered as 1. I do not understand other "relative quantifications" thoroughly in the manuscript (e.g., Fig 3G), as they are not relativized. An option would be to remove "relative" from the y-axis in the not relativized ones.

Fig 5D: The quantification of the TUNEL assay should be presented as a percentage of positive cells relative to the total number of cells (positive and negative), not just the total number of positives.

 

In general, I understand the effort put into this manuscript, but I recommend that it be carefully read and checked for minor details that need to be changed. Although the experimental level is acceptable, the presentation of data, including graphs and quantifications, should be done with care.

Author Response

Thank you very much for reviewing my manuscript and suggesting advice for revision. I have added experiments and modified the article according to your suggestions. Here is my point-to-point response to your question.

Lines 46-47: After a cell enters meiosis, it is not prone to DSBs, as there are programmed DSBs carried out by Spo11. Please clarify this sentence: Are there continuously occurring DSBs by SPO-11, or an accumulation of DNA breaks? Does this result in inhibited meiosis or failure of successful meiosis? Although there are mutations that lead to a massive accumulation of breaks, meiosis is still able to finish, although the oocyte may not be viable.

Thank you very much for your suggestion. I have revised this sentence. There was a mistake in my expression before. I have read the reference carefully, and this part has been changed to: After a cell enters mitosis, it is prone to different types of damage, such as DNA dou-ble-strand breaks. When DNA DSBs occurs during mitosis, the cell will continue to divide without DNA damage repair, so the wrong chromatids are expressed, preventing normal cell division and proliferation. Thanks again for your advice.

Lines 53-54: Reference 10 does not support this statement. Reference 10 discusses mouse meiosis. Please revise this sentence.

Thank you very much for your question. I'm sorry for my mistake. I have reinserted the correct references.

Line 54: Please (again) use Caenorhabditis elegans (in italics) and SPO-11 (not Spo-11).

Thank you very much for your advice. I have corrected the above errors.

Lines 57-58: Please revise this sentence.

Thank you very much for your suggestion. I have modified this sentence to : Jose Antonio et al. found that DNA damage caused by environmental influences led to the cell cycle to stop. In this way, the damaged DNA was prevented from replicating furthe. Thanks again for your advice.

Line 60: Please use "cell cycle progression" instead of "process".

Thank you very much for your advice. I have corrected this error.

Line 239: A P value < 0.01 indicates a more significant difference than a P value < 0.05.

Thank you very much for your suggestion. I have modified this sentence.

Figure 4, page 13: Please provide Figure legends. In panel c, what do the black and grey bars represent?

Thank you very much for your suggestion. The black bars represented NC and the grey bars represented siKDM5B. I have added the Figure legends into Figure 4.

A consideration for all the figures: In Fig 4C, 5B, and 5C, quantification is relative. For example, the protein levels in grey in Fig 4C are referred to as levels of the black ones, which are considered as 1. I do not understand other "relative quantifications" thoroughly in the manuscript (e.g., Fig 3G), as they are not relativized. An option would be to remove "relative" from the y-axis in the not relativized ones.

Thank you very much for your suggestion. I have modified the chart in the whole article. Change ‘Relative mRNA expression’ to ‘The mRNA expression’. Change ‘Relative protein expression’ to ‘The protein expression’. Thanks again for your advice.

Fig 5D: The quantification of the TUNEL assay should be presented as a percentage of positive cells relative to the total number of cells (positive and negative), not just the total number of positives.

Thank you very much for your suggestion. Since Hoechst staining was not performed in the TUNEL experiment before, the total number of cells and the proportion of apoptotic cells could not be counted. Therefore, I supplemented the TUNEL experiment after interference with KDM5B, and calculated the proportion of apoptotic cells. The new experimental results have been added to Fig 5D. Thanks again for your advice.