Reviewer ’s report  
 
Title: Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic 
steatohepatitis using a high -fat, choline -deficient diet and intraperitoneal injection of 
diethylnitrosamine  
 
 
Version: 0 Date: 17 Oct 2015  
 
 
Reviewer: Atsushi  Nakajima  
 
 
Reviewer's report:  
 
Norihiro K,  et al. developed a mouse model of hepatocellular carcinoma with NASH by using 
HFCD and one -time intraperitoneal injection of DEN. This model develop hepatocellular 
carcinomas from 20 weeks and have all clinically relevant features of NASH. This manuscript  is 
very interesting and have sufficient quality to be published in this journal.  
 
1. Why did the authors modify the concentration in the DEN drinking experiment? It was 
considered that 135mg/L was too toxic. The authors should explain about it.  
 
2. The a uthors exhibit not only NAS but also fibrosis stage in table 3.  
 
3. In methods, the authors should mention how they measured tumor number and size in the 
method.  
 
4. Was this model have liver cirrhosis? How much ratio was it?  
 
5. The authors should correct f igure2 -D. 
 
6. I n figure 4, the authors should explain the difference between MF and MF + DEN in 
more detail.  
 
Are the methods appropriate and well described?  
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 spec ify 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 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' below.  
 
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 n amed 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 i ncluded 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: Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic 
steatohepatitis using a high -fat, choline -deficient diet and intraperitoneal injection of 
diethylnitrosamine  
 
 
Version: 0 Date: 17 Oct 2015  
 
 
Reviewer: Atsushi  Nakajima  
 
 
Reviewer's report:  
 
Norihiro K,  et al. developed a mouse model of hepatocellular carcinoma with NASH by using 
HFCD and one -time intraperitoneal injection of DEN. This model develop hepatocellular 
carcinomas from 20 weeks and have all clinically relevant features of NASH. This manuscript  is 
very interesting and have sufficient quality to be published in this journal.  
 
1. Why did the authors modify the concentration in the DEN drinking experiment? It was 
considered that 135mg/L was too toxic. The authors should explain about it.  
 
2. The a uthors exhibit not only NAS but also fibrosis stage in table 3.  
 
3. In methods, the authors should mention how they measured tumor number and size in the 
method.  
 
4. Was this model have liver cirrhosis? How much ratio was it?  
 
5. The authors should correct f igure2 -D. 
 
6. I n figure 4, the authors should explain the difference between MF and MF + DEN in 
more detail.  
 
Are the methods appropriate and well described?  
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 spec ify 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 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' below.  
 
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 n amed 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 i ncluded 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: Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic 
steatohepatitis using a high -fat, choline -deficient diet and intraperitoneal injection of 
diethylnitrosamine  
 
 
Version: 0 Date: 17 Oct 2015  
 
 
Reviewer: Atsushi  Nakajima  
 
 
Reviewer's report:  
 
Norihiro K,  et al. developed a mouse model of hepatocellular carcinoma with NASH by using 
HFCD and one -time intraperitoneal injection of DEN. This model develop hepatocellular 
carcinomas from 20 weeks and have all clinically relevant features of NASH. This manuscript  is 
very interesting and have sufficient quality to be published in this journal.  
 
1. Why did the authors modify the concentration in the DEN drinking experiment? It was 
considered that 135mg/L was too toxic. The authors should explain about it.  
 
2. The a uthors exhibit not only NAS but also fibrosis stage in table 3.  
 
3. In methods, the authors should mention how they measured tumor number and size in the 
method.  
 
4. Was this model have liver cirrhosis? How much ratio was it?  
 
5. The authors should correct f igure2 -D. 
 
6. I n figure 4, the authors should explain the difference between MF and MF + DEN in 
more detail.  
 
Are the methods appropriate and well described?  
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 spec ify 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 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' below.  
 
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 n amed 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 i ncluded as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant