Reviewer ’s report  
 
Title: Releva nce of FXR -p62/SQSTM1 pathway for survival and protection of mouse 
hepatocytes and liver, especially with steatosis.  
 
Version: 0 Date: 03 Se p 2016  
 
Reviewer: Robert Schierwagen  
 
Reviewer's report:  
 
With their current work Haga et al. describe the importance of FXR -p62 signaling for 
antioxidative response and cell survival of hepatocytes  with regard to liver cell regeneration after 
partial hepatectomy, thereby extending their previous work on p62. They demonstrate an FXR 
dependent upregulation of p62 leading to increased antioxidant properties and survival in 
hepatocyte cell line AML12. I n vivo, FXR stimulation improves liver regeneration after partial 
hepatectomy in a murine model of steatosis. Nevertheless, the authors could do some 
improvements.  
 
Major comments  
1. In their initial experiments the authors tested different doses of FXR a gonist GW4064 and 
their effect on p62 expression in AML12 cells. Gene and protein expression of p62 were 
highest after treatment with 2 µM GW4064, while 0.5 or 1 µM of GW4064 failed to increase 
p62 to a significantly. However, in the following in vitro exp eriments the authors seem to 
favour treatment of cells with 1 µM GW4064. Consequently, treatment with 1 µM GW4064 
without addition of siRNA failed to increase p62 or Nrf2 expression. Again, in figure 2 and 
the corresponding results, highest effects on Nrf2  and pAkt are shown after treatment with 2 
µM GW4064, corresponding to figure 1. The authors should perform experiments with 2µM 
GW4064, where missing or at least discuss appropriately why they have chosen to prefer 
treatment with 1µM GW4064.  
2. In figure  2B, the effects of GW4064 treatment on antioxidant molecules Catalase, HO -1 and 
MnSOD can hardly be observed in the presented blots, especially because GAPDH levels 
seem to be higher in the treatment groups compared to the untreated control. The authors 
should provide quantifications of all western blots, like in figure 1, to strengthen their 
message.  
3. In the results the authors describe that FXR stimulation did not induce cell proliferation. Xie 
et al. (Sci Rep, 2016) reported that FXR activation induc ed proliferation in HepG2 cells. This 
discrepancy should be discussed accordingly.   
4. The discussion section should be revised since it mainly repeats the results and poorly 
integrate the work into the context of the existing research in the field. This  was done at least 
partly in the results section and those references and discussions should be done in the 
discussions section consistently. Possible other points for discussion are given in the above 
comments. Additional points are for example the oncoge nic properties of p62 (reviewed in 
Taniguchi et al., FEBS Lett., 2016), which could be a limitation for the study.  
 
Minor comments  
1. According to the author guidelines of BMC Gastroenterology references should not be cited 
in the abstract. Therefore, th e reference on page 2 line 7 should be removed.  
2. "PVDF" (page 10, line 8) should be written out  
3. In the results to figure 3A the authors described that the most effective concentration of 
GW4064 was 1 µM. Again, treatment with 2 µM of GW4064 was not shown in figure 3A, 
which showed most significant effects in figures 1 and 2. In figure 3B, the authors should 
show a western blot for GAPDH as in the other siRNA experiments in this study.  
4. In figure 4A it would be nice to show results from triglyceride content without T0901317 and 
GW4064 stimulation as a control and to demonstrate the effects of T0901317 stimulation and 
consequent GW4064 treatment.  
5. In figure 6 the overlay of the text box "Nrf2/ARE activation by binding to Keap -1" and the 
green arrow should be revised.  
Are the methods appropriate and well descr ibed?  
If not, please specify what is required in your comments 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 require 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:  
 
Acceptable  
 
Declaration of competing 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 from 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 futu re? 
 
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 your 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 ac cepted 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://creativecommo ns.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: Releva nce of FXR -p62/SQSTM1 pathway for survival and protection of mouse 
hepatocytes and liver, especially with steatosis.  
 
Version: 0 Date: 03 Se p 2016  
 
Reviewer: Robert Schierwagen  
 
Reviewer's report:  
 
With their current work Haga et al. describe the importance of FXR -p62 signaling for 
antioxidative response and cell survival of hepatocytes  with regard to liver cell regeneration after 
partial hepatectomy, thereby extending their previous work on p62. They demonstrate an FXR 
dependent upregulation of p62 leading to increased antioxidant properties and survival in 
hepatocyte cell line AML12. I n vivo, FXR stimulation improves liver regeneration after partial 
hepatectomy in a murine model of steatosis. Nevertheless, the authors could do some 
improvements.  
 
Major comments  
1. In their initial experiments the authors tested different doses of FXR a gonist GW4064 and 
their effect on p62 expression in AML12 cells. Gene and protein expression of p62 were 
highest after treatment with 2 µM GW4064, while 0.5 or 1 µM of GW4064 failed to increase 
p62 to a significantly. However, in the following in vitro exp eriments the authors seem to 
favour treatment of cells with 1 µM GW4064. Consequently, treatment with 1 µM GW4064 
without addition of siRNA failed to increase p62 or Nrf2 expression. Again, in figure 2 and 
the corresponding results, highest effects on Nrf2  and pAkt are shown after treatment with 2 
µM GW4064, corresponding to figure 1. The authors should perform experiments with 2µM 
GW4064, where missing or at least discuss appropriately why they have chosen to prefer 
treatment with 1µM GW4064.  
2. In figure  2B, the effects of GW4064 treatment on antioxidant molecules Catalase, HO -1 and 
MnSOD can hardly be observed in the presented blots, especially because GAPDH levels 
seem to be higher in the treatment groups compared to the untreated control. The authors 
should provide quantifications of all western blots, like in figure 1, to strengthen their 
message.  
3. In the results the authors describe that FXR stimulation did not induce cell proliferation. Xie 
et al. (Sci Rep, 2016) reported that FXR activation induc ed proliferation in HepG2 cells. This 
discrepancy should be discussed accordingly.   
4. The discussion section should be revised since it mainly repeats the results and poorly 
integrate the work into the context of the existing research in the field. This  was done at least 
partly in the results section and those references and discussions should be done in the 
discussions section consistently. Possible other points for discussion are given in the above 
comments. Additional points are for example the oncoge nic properties of p62 (reviewed in 
Taniguchi et al., FEBS Lett., 2016), which could be a limitation for the study.  
 
Minor comments  
1. According to the author guidelines of BMC Gastroenterology references should not be cited 
in the abstract. Therefore, th e reference on page 2 line 7 should be removed.  
2. "PVDF" (page 10, line 8) should be written out  
3. In the results to figure 3A the authors described that the most effective concentration of 
GW4064 was 1 µM. Again, treatment with 2 µM of GW4064 was not shown in figure 3A, 
which showed most significant effects in figures 1 and 2. In figure 3B, the authors should 
show a western blot for GAPDH as in the other siRNA experiments in this study.  
4. In figure 4A it would be nice to show results from triglyceride content without T0901317 and 
GW4064 stimulation as a control and to demonstrate the effects of T0901317 stimulation and 
consequent GW4064 treatment.  
5. In figure 6 the overlay of the text box "Nrf2/ARE activation by binding to Keap -1" and the 
green arrow should be revised.  
Are the methods appropriate and well descr ibed?  
If not, please specify what is required in your comments 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 require 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:  
 
Acceptable  
 
Declaration of competing 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 from 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 futu re? 
 
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 your 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 ac cepted 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://creativecommo ns.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: Releva nce of FXR -p62/SQSTM1 pathway for survival and protection of mouse 
hepatocytes and liver, especially with steatosis.  
 
Version: 0 Date: 03 Se p 2016  
 
Reviewer: Robert Schierwagen  
 
Reviewer's report:  
 
With their current work Haga et al. describe the importance of FXR -p62 signaling for 
antioxidative response and cell survival of hepatocytes  with regard to liver cell regeneration after 
partial hepatectomy, thereby extending their previous work on p62. They demonstrate an FXR 
dependent upregulation of p62 leading to increased antioxidant properties and survival in 
hepatocyte cell line AML12. I n vivo, FXR stimulation improves liver regeneration after partial 
hepatectomy in a murine model of steatosis. Nevertheless, the authors could do some 
improvements.  
 
Major comments  
1. In their initial experiments the authors tested different doses of FXR a gonist GW4064 and 
their effect on p62 expression in AML12 cells. Gene and protein expression of p62 were 
highest after treatment with 2 µM GW4064, while 0.5 or 1 µM of GW4064 failed to increase 
p62 to a significantly. However, in the following in vitro exp eriments the authors seem to 
favour treatment of cells with 1 µM GW4064. Consequently, treatment with 1 µM GW4064 
without addition of siRNA failed to increase p62 or Nrf2 expression. Again, in figure 2 and 
the corresponding results, highest effects on Nrf2  and pAkt are shown after treatment with 2 
µM GW4064, corresponding to figure 1. The authors should perform experiments with 2µM 
GW4064, where missing or at least discuss appropriately why they have chosen to prefer 
treatment with 1µM GW4064.  
2. In figure  2B, the effects of GW4064 treatment on antioxidant molecules Catalase, HO -1 and 
MnSOD can hardly be observed in the presented blots, especially because GAPDH levels 
seem to be higher in the treatment groups compared to the untreated control. The authors 
should provide quantifications of all western blots, like in figure 1, to strengthen their 
message.  
3. In the results the authors describe that FXR stimulation did not induce cell proliferation. Xie 
et al. (Sci Rep, 2016) reported that FXR activation induc ed proliferation in HepG2 cells. This 
discrepancy should be discussed accordingly.   
4. The discussion section should be revised since it mainly repeats the results and poorly 
integrate the work into the context of the existing research in the field. This  was done at least 
partly in the results section and those references and discussions should be done in the 
discussions section consistently. Possible other points for discussion are given in the above 
comments. Additional points are for example the oncoge nic properties of p62 (reviewed in 
Taniguchi et al., FEBS Lett., 2016), which could be a limitation for the study.  
 
Minor comments  
1. According to the author guidelines of BMC Gastroenterology references should not be cited 
in the abstract. Therefore, th e reference on page 2 line 7 should be removed.  
2. "PVDF" (page 10, line 8) should be written out  
3. In the results to figure 3A the authors described that the most effective concentration of 
GW4064 was 1 µM. Again, treatment with 2 µM of GW4064 was not shown in figure 3A, 
which showed most significant effects in figures 1 and 2. In figure 3B, the authors should 
show a western blot for GAPDH as in the other siRNA experiments in this study.  
4. In figure 4A it would be nice to show results from triglyceride content without T0901317 and 
GW4064 stimulation as a control and to demonstrate the effects of T0901317 stimulation and 
consequent GW4064 treatment.  
5. In figure 6 the overlay of the text box "Nrf2/ARE activation by binding to Keap -1" and the 
green arrow should be revised.  
Are the methods appropriate and well descr ibed?  
If not, please specify what is required in your comments 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 require 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:  
 
Acceptable  
 
Declaration of competing 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 from 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 futu re? 
 
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 your 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 ac cepted 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://creativecommo ns.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