Changes in the Cell Division of Chang Liver Cells Induced by Simulated Microgravity

Round 1

Reviewer 1 Report

The authors presented their work on the cell division under simulated microgravity. The work is interesting, with detailed experiments. However, the manuscript needs further polishing before I recommend it for publication. Here are some comments:

1.  The first 3 sentences show the similar meaning, can be simplified. And the introduction should be rewritten with a clearer logic.

2. There are small mistakes, i.e. line 55, page 2, before and after [11], there is a dot; there are two dots at the end of Introduction, etc.

3. Line 73, page 2, “…. incubated at 37 ^oC in water bath” for how long? Line 76, “The cells were split…”, The cells with or without culture medium?

4. The culture process appeared several times, which is not necessary.

5. Line 110, page 3, what is missed after total?

6. Line 123, page 3, “Following are the steps taken for the thermal cycle:…”, it is not friendly reading, separating from ; with Enter will be better.

7. Section 2.5, the title is “microtubule staining”, but nothing related was introduced in this section, instead, the culture process which was already presented in previous sections is shown again.

8. Figure 1b, the texts in the figure are unreadable. Same problem for Fig. 2e.

9. Why "***" and "** can represent the "significant different" at the same time?

10. There is no “immunofluorescence staining” in methods.

Author Response

Dear Prof. Dr. Takayoshi Kobayashi, Editorial Boards and Reviewers,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

1. The first 3 sentences show the similar meaning, can be simplified. And the introduction should be rewritten with a clearer logic.

Authors’ response:

The authors have simplified these sentences in the manuscript.

2. There are small mistakes, i.e. line 55, page 2, before and after [11], there is a dot; there are two dots at the end of Introduction, etc.

Authors’ response:

The authors have corrected these mistakes.

3. Line 73, page 2, “…. incubated at 37 ^oC in water bath” for how long? Line 76, “The cells were split…”, The cells with or without culture medium?

Authors’ responses:

Line 73: The cells were incubated for 30 s (added to the manuscript).

Line 76: The authors have changed this sentence to “The cells were transferred...”. We mentioned in the previous sentence: “The medium was removed and the cell pellet was mixed with 1 mL cell culture medium”, indicated that the old medium was removed and the cells was mixed with new medium, then the cell with medium were transferred to T-25 flask.

4. The culture process appeared several times, which is not necessary.

Authors’ response: In Materials and Methods, we used diferent methods to access the characteristics of  CCL-13 cells. Each method uses a different number of cells (1 × 10⁵ or 2 × 10³ cells) and different culture equipment (T-25 tissue culture flask  or 96-well microplates). Therefore, we want to clarify the cell culture process in each method so that it is easy for readers to access.

5. Line 110, page 3, what is missed after total?

Authors’ response:

The authors have edited to “…total RNA”.

6. Line 123, page 3, “Following are the steps taken for the thermal cycle:…”, it is not friendly reading, separating from ; with Enter will be better.

Authors’ response: the “Following are the steps taken for the thermal cycle:…” was corrected to “The thermal cycle were performed by”

7. Section 2.5, the title is “microtubule staining”, but nothing related was introduced in this section, instead, the culture process which was already presented in previous sections is shown again.

Authors’ response: in this method, microtubule struture of CCL-13 cells was evaluated by staining with SiR-tubulin (CY-SC002, Cytoskeleton, Inc., United States). 50 nM SiR-tubulin/well was used to label microtubules of CCL-13 cells for 72 h and then directly observed under flourescent microscope. 

8. Figure 1b, the texts in the figure are unreadable. Same problem for Fig. 2e.

Authors’ response: we have corrected the text in these Figure.

9. Why "***" and "** can represent the "significant different" at the same time?

Authors’ response:

In Figure 1, we compared the difference within each phase of CCL-13 cells. "***" represent the significant different (p<0.001) between control and SMG groups of CCL-13 cells going to G2/M phase, and "**" represent the significant different (p<0.01) between control and SMG groups of these cells going to G0/G1(or S) phase.

10. There is no “immunofluorescence staining” in methods.

Authors’ response: The authors have corrected “immunofluorescence staining” to “microfilament and microtubule staining” in the section 3.4 of the Results.

We hope that our corrections could meet you requirements,

Thank you so much.

 

Reviewer 2 Report

Dear editor,  

In this manuscript titled ‘The changes in the cell division of Chang liver cells induced by 
simulated microgravity’, the authors (Minh Thi Tran and colleagues) examined the changes in cell division in Chang liver cells (CCL-13 cells) under simulated microgravity conditions. The work is not novel as other research groups already proved that microgravity can affect cell division (Peng Wang et al., FASEB J, 2019). My view is that this paper does not contribute significantly to the fields of cell division and cell cycle. The story did not go into details of cell division. Hence the manuscript is not suitable for publication in the present format. My concerns and comments are mentioned below.  

 

• Introduction needs to be more detailed where authors should discuss simulated microgravity (SMG) and the cell division aspects including chromosome condensation, spindle structures and/or kinetochore structures why they want to aim to study them. 

 

• Figure 1: Result section 3.1 should be rewritten. How many times did they run the experiment? How many cells did the authors analyze? I am asking this as the differences between control and SMG samples are minimal even though they are statistically significant.  

 

• Figure 2: The differences between control and SMG treated samples were minimal, especially in nuclear area and nuclear intensities. Authors should comment on them.  

 

• Figure 3 and 4: The assays were nicely performed, and the observations and conclusions were clear. For figure 4, labeling can be improved. Authors should mention counts of cells in which they noticed the phenotype. 

 

• Figure 5: I am confused about what they want to convey here. The labelling of the figure should be improved significantly.  

 

• Authors did not show the expressions (protein/transcripts) of components of centromere and kinetochore and that of spindle assembly checkpoint.  

 

• They did not investigate or even comment on the G0-G1 population cells of SMG treated sample if there is any sort of cell cycle checkpoint become activated.  

 

• Did SMG trigger apoptosis or some kind of cell death mechanism? I am asking this as in figure 1, SMG treated cells showed some sub-G1 population, the amount of which was more than what they observed in control cells.

Author Response

Dear Prof. Dr. Takayoshi Kobayashi, Editorial Boards and Reviewers,

 

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are in the attached file.

 

We hope that our corrections could meet your requirements,

 

Thank you so much.

Best regards,

 

Author Response File: Author Response.docx

Reviewer 3 Report

A study by Tran et al focusing on the effect of SM on liver cells with respect to cell division presents relatively good quality od data. To strengthen the conclusions, the following comments should be addressed.

Comments: 

1) please correct English.

2) please define scale bars in every figures containing microphotographs.

3) expression of cell cycle-related genes should be determined at the protein level.

The Authors refer to relevant literature and provide sufficient discussion.

Author Response

Dear Prof. Dr. Takayoshi Kobayashi, Editorial Boards and Reviewers,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

1) please correct English.

Authors’ response: The authors have corrected English mistakes in the revision.

2) please define scale bars in every figures containing microphotographs.

Authors’ response: we have define scale bars for the figures in our manuscript

3) expression of cell cycle-related genes should be determined at the protein level.

Authors’ response: We added the evaluation of the protein expression of these genes in Figure 4 (Results).

We hope that our corrections could meet your requirements,

Thank you so much,

Best regards,

Reviewer 4 Report

The author conducted to determine how CCL-13 cells’ division has changed under simulated microgravity (SMG), demonstrating by the alteration in the cell cycle, the changes in cell area and nuclear area, the nuclear/cytoplasmic ratio, and the transcript expression of genes associated with the cell cycle. They also estimate the morphology of divided cells, such as cell shape and microvilli appearance, and assess the cleavage furrow formation and polar spindle microtubules contributing to cell division during cytokinesis. Albeit, I consider these findings to provide new insight into SMG-related fields, I still have some suggestions.

1, Most figures and tables are highly professional; however, the authors should guide the readers to the meaning of the images and tables appropriately; otherwise, it will likely cause misunderstandings. Therefore, I suggest the author consider revising these figure legends and tables again.

2, The author revealed SMG attenuated the cell division of CCL-13 cells by driving cells to the arrest phase and altering the cell division structures. However, it would be much better if the authors could provide some Workflow or Scheme for this research, I suggest that they can take a look at the recent paper in MDPI (PMID:  35563422, 35645371, 33371243)

3, In some figures, the author presented the bar graph of statistical analysis. However, the author may need to use other statistical analyses, such as ANOVA to calculate the P-value for three or more groups of data, and please update the “Statistical Analysis” of the Method during further revision(PMID: 37114505, 36555654)

4, There are few typo issues for the authors to pay attention to; Please also unify the writing of scientific terms. “Italic, capital”?

 

5, The font is too small for some of the current figures; meanwhile, the manuscript also needs English proofreading.

Author Response

Dear Prof. Dr. Takayoshi Kobayashi, Editorial Boards and Reviewers,

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

1, Most figures and tables are highly professional; however, the authors should guide the readers to the meaning of the images and tables appropriately; otherwise, it will likely cause misunderstandings. Therefore, I suggest the author consider revising these figure legends and tables again.

Authors’ response: We have corrected and adjusted the Figures and the legends.

2, The author revealed SMG attenuated the cell division of CCL-13 cells by driving cells to the arrest phase and altering the cell division structures. However, it would be much better if the authors could provide some Workflow or Scheme for this research, I suggest that they can take a look at the recent paper in MDPI (PMID:  35563422, 35645371, 33371243)

Authors’ response: We have added the schematic representation of the experimental design for our manuscript (Figure 1).

3, In some figures, the author presented the bar graph of statistical analysis. However, the author may need to use other statistical analyses, such as ANOVA to calculate the P-value for three or more groups of data, and please update the “Statistical Analysis” of the Method during further revision(PMID: 37114505, 36555654)

Authors’ response: Thank you so much for your comment, this is our mistake during prepare this manuscript. “One way ANOVA” was corrected to “Holm-Sidak test” in the statistical analyses.

4, There are few typo issues for the authors to pay attention to; Please also unify the writing of scientific terms. “Italic, capital”?

Authors’ response:

The authors have corrected gene terms as following:

• cyclin B1 and cyclin D1 are full names of genes: not italicize
• CDK2 and CDK6 are gene symbols: Italic, capital

5, The font is too small for some of the current figures; meanwhile, the manuscript also needs English proofreading.

Authors’ response:

The authors have edited these figures and improved the English of the manuscript.

We hope that our corrections could meet your requirements,

Thank you so much.

Round 2

Reviewer 2 Report

Dear editor, I have read the manuscript in its present form and my vote is to accept it for publication. I have no more comments or suggestions. 

Reviewer 3 Report

Comments have been addressed.