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: 18 Aug  2016  
 
Reviewer: Pamela Tuma  
 
Reviewer's report:  
 
The authors pose a very interesting scenario of FXR agonist -stimulated hepatoprotection in 
steatosis via p62/SQSTM1 -mediated signaling to activate survival (via Nrf2) and suppress 
steatosis (via SHP).  In general the approach is fine and all the relevant m olecules were 
examined.  However, in this reviewer's opinion, many of the conclusions drawn by the authors 
on changes in protein expression levels, cell survival and hepatoprotection are overstated, very 
modest or simply not convincing thereby diminishing the enthusiasm for this manuscript.  This 
concern and others are listed below in no particular order.  
Many of the conclusions concerning changes in protein expression levels are modest or not 
convincing.  This is in large part due to the wide variability in GAPDH levels (the loading 
control) detected among samples.  Because the authors state they loaded 25 ug protein/lane, one 
would expect much more even GAPDH detection (see Fig. 1A - also is that determination from 
an n = 1 only?).  In many cases, when "e yeballed" for normalization to GAPDH levels, changes 
in the expression levels of the proteins of interest would be negated.  For example, in Fig. 2, the 
negative control in the first 2 lanes has less GAPDH than in the remaining lanes from which the 
increas ed expression levels were concluded.  Also, many of the duplicate samples for the 
proteins of interest vary widely while the GAPDH levels are consistent for the given pair.  
Careful densitometry and normalization are needed to confirm the conclusions made.   
Also, the knockdown for p62 and GAPDH is modest at best (see Fig. 1C) thereby making firm 
conclusions difficult.  Similarly, changes in cell survival are modest (Fig. 3) and it is not clear 
whether the small increases are due to enhanced proliferation o r decreased cell death.  Are these 
modest differences physiologically relevant - especially given the modest improvement in liver 
injury in agonist -treated mice?  
The methods section was missing many experimental details and/or references to specific 
proto cols.  For example, no citations or experimental protocols were provided for the animal 
procedures or the Sudan III labeling of the cells.  Also, details of the antibodies used for 
immunoblotting were missing (e.g., dilutions, incubations, secondaries, etc .).  It would be 
difficult to reproduce these studies - which is a major concern in this climate where 
reproducibility in science has become a big conversation.  
The figure legends were also lacking details.  For example, are duplicates loaded on the 
immun oblots in Figs. 2, 4 and 5?  Are they duplicates taken from the same dish of cells or from 
independent experiments performed at different times?  Do they represent different animals in 
Fig. 5D?  The lack of detail makes it somewhat difficult to be certain that I am interpreting the 
data correctly thereby precluding a firm assessment of the findings.   
Is it possible that the wrong model systems have been used for these studies?  Are the increased 
levels of TGF -alpha produced by the AML12 cells complicating  the results?  Are the leptin 
receptor -deficient (db/db) mice too extreme of a model for steatosis?  It might be useful to 
discuss the limitations of the models used to help explain the modest effects observed in agonist -
stimulated cells/animals.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional stat istical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in t he 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 organisat ion 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 reimbursemen ts, 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 t his 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 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 comme nts 
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: 18 Aug  2016  
 
Reviewer: Pamela Tuma  
 
Reviewer's report:  
 
The authors pose a very interesting scenario of FXR agonist -stimulated hepatoprotection in 
steatosis via p62/SQSTM1 -mediated signaling to activate survival (via Nrf2) and suppress 
steatosis (via SHP).  In general the approach is fine and all the relevant m olecules were 
examined.  However, in this reviewer's opinion, many of the conclusions drawn by the authors 
on changes in protein expression levels, cell survival and hepatoprotection are overstated, very 
modest or simply not convincing thereby diminishing the enthusiasm for this manuscript.  This 
concern and others are listed below in no particular order.  
Many of the conclusions concerning changes in protein expression levels are modest or not 
convincing.  This is in large part due to the wide variability in GAPDH levels (the loading 
control) detected among samples.  Because the authors state they loaded 25 ug protein/lane, one 
would expect much more even GAPDH detection (see Fig. 1A - also is that determination from 
an n = 1 only?).  In many cases, when "e yeballed" for normalization to GAPDH levels, changes 
in the expression levels of the proteins of interest would be negated.  For example, in Fig. 2, the 
negative control in the first 2 lanes has less GAPDH than in the remaining lanes from which the 
increas ed expression levels were concluded.  Also, many of the duplicate samples for the 
proteins of interest vary widely while the GAPDH levels are consistent for the given pair.  
Careful densitometry and normalization are needed to confirm the conclusions made.   
Also, the knockdown for p62 and GAPDH is modest at best (see Fig. 1C) thereby making firm 
conclusions difficult.  Similarly, changes in cell survival are modest (Fig. 3) and it is not clear 
whether the small increases are due to enhanced proliferation o r decreased cell death.  Are these 
modest differences physiologically relevant - especially given the modest improvement in liver 
injury in agonist -treated mice?  
The methods section was missing many experimental details and/or references to specific 
proto cols.  For example, no citations or experimental protocols were provided for the animal 
procedures or the Sudan III labeling of the cells.  Also, details of the antibodies used for 
immunoblotting were missing (e.g., dilutions, incubations, secondaries, etc .).  It would be 
difficult to reproduce these studies - which is a major concern in this climate where 
reproducibility in science has become a big conversation.  
The figure legends were also lacking details.  For example, are duplicates loaded on the 
immun oblots in Figs. 2, 4 and 5?  Are they duplicates taken from the same dish of cells or from 
independent experiments performed at different times?  Do they represent different animals in 
Fig. 5D?  The lack of detail makes it somewhat difficult to be certain that I am interpreting the 
data correctly thereby precluding a firm assessment of the findings.   
Is it possible that the wrong model systems have been used for these studies?  Are the increased 
levels of TGF -alpha produced by the AML12 cells complicating  the results?  Are the leptin 
receptor -deficient (db/db) mice too extreme of a model for steatosis?  It might be useful to 
discuss the limitations of the models used to help explain the modest effects observed in agonist -
stimulated cells/animals.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional stat istical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in t he 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 organisat ion 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 reimbursemen ts, 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 t his 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 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 comme nts 
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: 18 Aug  2016  
 
Reviewer: Pamela Tuma  
 
Reviewer's report:  
 
The authors pose a very interesting scenario of FXR agonist -stimulated hepatoprotection in 
steatosis via p62/SQSTM1 -mediated signaling to activate survival (via Nrf2) and suppress 
steatosis (via SHP).  In general the approach is fine and all the relevant m olecules were 
examined.  However, in this reviewer's opinion, many of the conclusions drawn by the authors 
on changes in protein expression levels, cell survival and hepatoprotection are overstated, very 
modest or simply not convincing thereby diminishing the enthusiasm for this manuscript.  This 
concern and others are listed below in no particular order.  
Many of the conclusions concerning changes in protein expression levels are modest or not 
convincing.  This is in large part due to the wide variability in GAPDH levels (the loading 
control) detected among samples.  Because the authors state they loaded 25 ug protein/lane, one 
would expect much more even GAPDH detection (see Fig. 1A - also is that determination from 
an n = 1 only?).  In many cases, when "e yeballed" for normalization to GAPDH levels, changes 
in the expression levels of the proteins of interest would be negated.  For example, in Fig. 2, the 
negative control in the first 2 lanes has less GAPDH than in the remaining lanes from which the 
increas ed expression levels were concluded.  Also, many of the duplicate samples for the 
proteins of interest vary widely while the GAPDH levels are consistent for the given pair.  
Careful densitometry and normalization are needed to confirm the conclusions made.   
Also, the knockdown for p62 and GAPDH is modest at best (see Fig. 1C) thereby making firm 
conclusions difficult.  Similarly, changes in cell survival are modest (Fig. 3) and it is not clear 
whether the small increases are due to enhanced proliferation o r decreased cell death.  Are these 
modest differences physiologically relevant - especially given the modest improvement in liver 
injury in agonist -treated mice?  
The methods section was missing many experimental details and/or references to specific 
proto cols.  For example, no citations or experimental protocols were provided for the animal 
procedures or the Sudan III labeling of the cells.  Also, details of the antibodies used for 
immunoblotting were missing (e.g., dilutions, incubations, secondaries, etc .).  It would be 
difficult to reproduce these studies - which is a major concern in this climate where 
reproducibility in science has become a big conversation.  
The figure legends were also lacking details.  For example, are duplicates loaded on the 
immun oblots in Figs. 2, 4 and 5?  Are they duplicates taken from the same dish of cells or from 
independent experiments performed at different times?  Do they represent different animals in 
Fig. 5D?  The lack of detail makes it somewhat difficult to be certain that I am interpreting the 
data correctly thereby precluding a firm assessment of the findings.   
Is it possible that the wrong model systems have been used for these studies?  Are the increased 
levels of TGF -alpha produced by the AML12 cells complicating  the results?  Are the leptin 
receptor -deficient (db/db) mice too extreme of a model for steatosis?  It might be useful to 
discuss the limitations of the models used to help explain the modest effects observed in agonist -
stimulated cells/animals.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional stat istical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in t he 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 organisat ion 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 reimbursemen ts, 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 t his 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 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 comme nts 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant