Reviewer’s report 
Title:  Niclosamide induced cell apoptosis via upregulatio n of ATF3 and activation of PERK in 
Hepatocellular carcinoma cells 
 
Version:  0 Date:  01 Nov 2015 
 
Reviewer:  Haibo Bai 
 
Reviewer's report: 
Niclosamide has been shown to induce apoptosis in m ultiple cancer cell lines in vitro and in vivo 
by inhibiting multiple oncogenic signaling pathways . In this manuscript, the authors showed that 
niclosamide induce ER stress and subsequently ER st ress-related apoptosis by upregulating 
PERK kinase activity transcriptionally as well as p ost-translationally, which subsequently 
upregulates ATF3, ATF4 and CHOP levels. This manusc ript identified a new mechanism of 
niclosamide-induced cancer cell apoptosis.  
 
Major comments: 
1. Since eIF2 α is directly phospohorylated by PERK upon ER stress  and is a major effector of 
ER stress. The phosphorylation levels of eIF2 α upon niclosamide should be measured to 
confirm the induction of ER stress.  
2. Salubrinal is not an inhibitor of PERK. It is a inhibitor of eIF2 α dephosphorylation, so it is 
considered as an inhibitor of ER stress. 
3. Figure 4, the IF pictures are not clear enough t o observe the nuclear accumulation of the 
proteins. The Quality of these pictures needs to be  improved.  
 
Minor comments:  
1. Figure 6B, it should be ATF3, not ATF5. Furtherm ore, it should be shRNA not RNAi. 
  
Are the methods appropriate and well described? 
If not, please specify what is required in your com ments 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 t he data shown? 
If not, please explain in your comments to the auth ors. 
Yes 
Are you able to assess any statistics in the manusc ript 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 manu script: 
Acceptable 
Declaration of competing interests 
Please complete a declaration of competing interest s, considering the following questions: 
1. Have you in the past five years received reimbur sements, fees, funding, or salary from an 
organisation that may in any way gain or lose finan cially 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 an y 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 intere sts? 
6. Do you have any non-financial competing interest s 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 det ails below. 
I declare that I have no competing interests 
I agree to the open peer review policy of the journ al. 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 o n the website along with the authors' 
responses. I agree for my report to be made availab le 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 repo rt can be included as confidential comments 
to the editors, which will not be published. 
I agree to the open peer review policy of the journ al 
Reviewer’s report 
Title:  Niclosamide induced cell apoptosis via upregulatio n of ATF3 and activation of PERK in 
Hepatocellular carcinoma cells 
 
Version:  0 Date:  01 Nov 2015 
 
Reviewer:  Haibo Bai 
 
Reviewer's report: 
Niclosamide has been shown to induce apoptosis in m ultiple cancer cell lines in vitro and in vivo 
by inhibiting multiple oncogenic signaling pathways . In this manuscript, the authors showed that 
niclosamide induce ER stress and subsequently ER st ress-related apoptosis by upregulating 
PERK kinase activity transcriptionally as well as p ost-translationally, which subsequently 
upregulates ATF3, ATF4 and CHOP levels. This manusc ript identified a new mechanism of 
niclosamide-induced cancer cell apoptosis.  
 
Major comments: 
1. Since eIF2 α is directly phospohorylated by PERK upon ER stress  and is a major effector of 
ER stress. The phosphorylation levels of eIF2 α upon niclosamide should be measured to 
confirm the induction of ER stress.  
2. Salubrinal is not an inhibitor of PERK. It is a inhibitor of eIF2 α dephosphorylation, so it is 
considered as an inhibitor of ER stress. 
3. Figure 4, the IF pictures are not clear enough t o observe the nuclear accumulation of the 
proteins. The Quality of these pictures needs to be  improved.  
 
Minor comments:  
1. Figure 6B, it should be ATF3, not ATF5. Furtherm ore, it should be shRNA not RNAi. 
  
Are the methods appropriate and well described? 
If not, please specify what is required in your com ments 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 t he data shown? 
If not, please explain in your comments to the auth ors. 
Yes 
Are you able to assess any statistics in the manusc ript 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 manu script: 
Acceptable 
Declaration of competing interests 
Please complete a declaration of competing interest s, considering the following questions: 
1. Have you in the past five years received reimbur sements, fees, funding, or salary from an 
organisation that may in any way gain or lose finan cially 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 an y 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 intere sts? 
6. Do you have any non-financial competing interest s 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 det ails below. 
I declare that I have no competing interests 
I agree to the open peer review policy of the journ al. 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 o n the website along with the authors' 
responses. I agree for my report to be made availab le 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 repo rt can be included as confidential comments 
to the editors, which will not be published. 
I agree to the open peer review policy of the journ al 
Please review this paper.
user
Reviewer’s report 
Title:  Niclosamide induced cell apoptosis via upregulatio n of ATF3 and activation of PERK in 
Hepatocellular carcinoma cells 
 
Version:  0 Date:  01 Nov 2015 
 
Reviewer:  Haibo Bai 
 
Reviewer's report: 
Niclosamide has been shown to induce apoptosis in m ultiple cancer cell lines in vitro and in vivo 
by inhibiting multiple oncogenic signaling pathways . In this manuscript, the authors showed that 
niclosamide induce ER stress and subsequently ER st ress-related apoptosis by upregulating 
PERK kinase activity transcriptionally as well as p ost-translationally, which subsequently 
upregulates ATF3, ATF4 and CHOP levels. This manusc ript identified a new mechanism of 
niclosamide-induced cancer cell apoptosis.  
 
Major comments: 
1. Since eIF2 α is directly phospohorylated by PERK upon ER stress  and is a major effector of 
ER stress. The phosphorylation levels of eIF2 α upon niclosamide should be measured to 
confirm the induction of ER stress.  
2. Salubrinal is not an inhibitor of PERK. It is a inhibitor of eIF2 α dephosphorylation, so it is 
considered as an inhibitor of ER stress. 
3. Figure 4, the IF pictures are not clear enough t o observe the nuclear accumulation of the 
proteins. The Quality of these pictures needs to be  improved.  
 
Minor comments:  
1. Figure 6B, it should be ATF3, not ATF5. Furtherm ore, it should be shRNA not RNAi. 
  
Are the methods appropriate and well described? 
If not, please specify what is required in your com ments 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 t he data shown? 
If not, please explain in your comments to the auth ors. 
Yes 
Are you able to assess any statistics in the manusc ript 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 manu script: 
Acceptable 
Declaration of competing interests 
Please complete a declaration of competing interest s, considering the following questions: 
1. Have you in the past five years received reimbur sements, fees, funding, or salary from an 
organisation that may in any way gain or lose finan cially 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 an y 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 intere sts? 
6. Do you have any non-financial competing interest s 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 det ails below. 
I declare that I have no competing interests 
I agree to the open peer review policy of the journ al. 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 o n the website along with the authors' 
responses. I agree for my report to be made availab le 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 repo rt can be included as confidential comments 
to the editors, which will not be published. 
I agree to the open peer review policy of the journ al 
assistant