Reviewer ’s report  
 
Title: Relevance of FXR -p62/SQSTM1 pathway for survival and protection of mouse 
hepatocytes and liver, especially with steatosis.  
 
Version: 0 Date: 23 Aug 2016  
 
Reviewer: Christopher L. Gentile  
 
Reviewer's report:  
 
Haga et al., examined the effects of the FXR agoinist, GW4064, in cultured hepatocytes and 
diabetic mice following hepatectomy. The authors found t hat GW4064 was protective, in part via 
a p62/SQSTM1 -dependent mechanism. The experiments appear well conducted. Several 
questions and concerns are listed below:  
1. As described by the authors, the experiments were performed because of the clinical 
releva nce of NAFLD and PH. However, all of the cell data (except for figure 4b) are 
performed in healthy cells with no insult. The data would be much more relevant to NAFLD 
if the cells were challenged with an insult that promotes NAFLD (e.g. long chain saturate d 
fatty acids) and the protective effects of GW4064 were examined.  
2. The authors talk extensively about cell survival/death, although the related measures are all 
indirect.  Figure 3 does describe cell survival via the xCelligence system, but it's uncle ar 
whether they are measuring viability, senescence, apoptosis, etc. The system should have the 
capability to distinguish among these processes. As mentioned above, it would be more 
relevant if these  outcomes were measured after an insult that caused cell  death, then the 
protective effects of GW4064 were examined.  
3. Regarding the western data, error bars are missing on several graphs (e.g. figure 1, 2), and it 
would be helpful to add bar graphs to figures 4 and 5 of the three separate experiments rather  
than just representative blots.  
4. Regarding figure 5a, there are no control data for the diabetic mice (e.g. sham or before 
surgery) to analyze the effects of diabetes, per se. Similarly, in figure 4B there are no 
triglyceride data without T0901317, th us no baseline data for comparison.  
Are the methods appropriate and well described?  
If not, please specify what is required in your c omments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects req uire further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competin g interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially fr om the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If you r reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/ licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Relevance of FXR -p62/SQSTM1 pathway for survival and protection of mouse 
hepatocytes and liver, especially with steatosis.  
 
Version: 0 Date: 23 Aug 2016  
 
Reviewer: Christopher L. Gentile  
 
Reviewer's report:  
 
Haga et al., examined the effects of the FXR agoinist, GW4064, in cultured hepatocytes and 
diabetic mice following hepatectomy. The authors found t hat GW4064 was protective, in part via 
a p62/SQSTM1 -dependent mechanism. The experiments appear well conducted. Several 
questions and concerns are listed below:  
1. As described by the authors, the experiments were performed because of the clinical 
releva nce of NAFLD and PH. However, all of the cell data (except for figure 4b) are 
performed in healthy cells with no insult. The data would be much more relevant to NAFLD 
if the cells were challenged with an insult that promotes NAFLD (e.g. long chain saturate d 
fatty acids) and the protective effects of GW4064 were examined.  
2. The authors talk extensively about cell survival/death, although the related measures are all 
indirect.  Figure 3 does describe cell survival via the xCelligence system, but it's uncle ar 
whether they are measuring viability, senescence, apoptosis, etc. The system should have the 
capability to distinguish among these processes. As mentioned above, it would be more 
relevant if these  outcomes were measured after an insult that caused cell  death, then the 
protective effects of GW4064 were examined.  
3. Regarding the western data, error bars are missing on several graphs (e.g. figure 1, 2), and it 
would be helpful to add bar graphs to figures 4 and 5 of the three separate experiments rather  
than just representative blots.  
4. Regarding figure 5a, there are no control data for the diabetic mice (e.g. sham or before 
surgery) to analyze the effects of diabetes, per se. Similarly, in figure 4B there are no 
triglyceride data without T0901317, th us no baseline data for comparison.  
Are the methods appropriate and well described?  
If not, please specify what is required in your c omments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects req uire further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competin g interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially fr om the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If you r reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/ licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Relevance of FXR -p62/SQSTM1 pathway for survival and protection of mouse 
hepatocytes and liver, especially with steatosis.  
 
Version: 0 Date: 23 Aug 2016  
 
Reviewer: Christopher L. Gentile  
 
Reviewer's report:  
 
Haga et al., examined the effects of the FXR agoinist, GW4064, in cultured hepatocytes and 
diabetic mice following hepatectomy. The authors found t hat GW4064 was protective, in part via 
a p62/SQSTM1 -dependent mechanism. The experiments appear well conducted. Several 
questions and concerns are listed below:  
1. As described by the authors, the experiments were performed because of the clinical 
releva nce of NAFLD and PH. However, all of the cell data (except for figure 4b) are 
performed in healthy cells with no insult. The data would be much more relevant to NAFLD 
if the cells were challenged with an insult that promotes NAFLD (e.g. long chain saturate d 
fatty acids) and the protective effects of GW4064 were examined.  
2. The authors talk extensively about cell survival/death, although the related measures are all 
indirect.  Figure 3 does describe cell survival via the xCelligence system, but it's uncle ar 
whether they are measuring viability, senescence, apoptosis, etc. The system should have the 
capability to distinguish among these processes. As mentioned above, it would be more 
relevant if these  outcomes were measured after an insult that caused cell  death, then the 
protective effects of GW4064 were examined.  
3. Regarding the western data, error bars are missing on several graphs (e.g. figure 1, 2), and it 
would be helpful to add bar graphs to figures 4 and 5 of the three separate experiments rather  
than just representative blots.  
4. Regarding figure 5a, there are no control data for the diabetic mice (e.g. sham or before 
surgery) to analyze the effects of diabetes, per se. Similarly, in figure 4B there are no 
triglyceride data without T0901317, th us no baseline data for comparison.  
Are the methods appropriate and well described?  
If not, please specify what is required in your c omments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects req uire further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competin g interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially fr om the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If you r reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/ licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant