Reviewer’s report 
Title:  Niclosamide induced cell apoptosis via upregulatio n of ATF3 and activation of PERK in 
Hepatocellular carcinoma cells 
 
Version:  0 Date:  16 Nov 2015 
 
Reviewer:  Syeda Afroze 
 
Reviewer's report: 
This manuscript entitled "Niclosamide induces cell apoptosis through upregulating ATF3 and 
activating PERK pathway in Hepatocellular carcinoma  cells" submitted by Weng  et al, the 
authors aimed to evaluate the effect of Niclosamide  on the Hepatocellular carcinoma cells. There 
are lots of issue to promote this hepatocellular ca rcinoma, such as HBV, HCV, Non-alcoholic 
fatty liver disease (NAFLD), Non-alcoholic steatohe patitis (NASH), Alcohol intake and 
Hemochromatosis as well. Hepatocellular carcinoma ( HCC) is one of the most devastating 
malignancy in human around the world. Above all the  prognosis of this disease is very much 
challenging. Even after the detection and taking ca re of it, the retrieval from this disease is very 
poor due to the lack of proper drugs (chemotherapeu tic) which has less or no side effect and the 
other reason gradually tumor cells tend to get chem o-resistance. That is why the survey for the 
new drugs for HCC treatment is necessary. Since Nic losamide is an antihelminthic drug which 
has been used to treat tapeworm infections also has  been shown anticancer effect in different 
tumor cell types as well as leukemia, colon cancer and glioma. Therefore in this study the 
authors assessed the anticancerous functional activ ity and mechanism of Niclosamide on two 
different HCC cell lines HepG2 and QGY7701. To expl ore the anti-HCC function and 
mechanism of Niclosamide in these two HCC cell line s, the authors performed several 
experiments such as, Cell viability assay, western blot, qPCR, Flow Cytometry and also did 
immunofluorescence in response to Niclosamide. The authors performed qPCR for evaluating 
these genes expressions such as, mRNA expression of  PERK, ATF3, ATF4 and CHOP, which 
are related to PERK signaling pathway. The authors also demonstrated western blot to see the 
phosphorylation of PERK (pPERK), protein expression  of caspase-3, PARP, PERK, CHOP, 
ATF3 and ATF4.The authors did Immunofluorescence st udy to see the location of expression of 
ATF3 and CHOP in these two HCC cell lines as well. The authors also conducted experiment by 
using PERK inhibitor GSK2606414 and knockdown the A TF3 gene to confirm that nicloamide 
is activating apoptosis through this PERK pathway v ia the upregulating ATF3 espression.It is an 
interesting study and work has done very well. But the presentation of this manuscript is not well 
organized. And also some certain aspects not well d escribed. So before going for final 
submission of this manuscript, it is recommended to  take care of this manuscript in terms of the 
following extensive major revision. Major concerns are mentioned below: 
 
Major Concerns: 
1. Title should be retitled. For an example, title could be "Niclosamide induced cell apoptosis 
via upregulation of ATF3 and activation of PERK in Hepatocellular carcinoma cells."   
2. The abstract and the manuscript should be carefu lly checked in terms of grammar. Need to 
revise by Scientific English editor. 
3. There are some information in the Background sec tion need to be removed from there and 
add it to the Materials and Methods section and res ult section as well. (Example: Last 
paragraph of the Background section "For investigat ing niclosamide anti-HCC function and 
mechanism, HepG2 and QGY7701 HCC cell lines…… , an inhibitor of PERK.) 
4. Before treating the cells with niclosamide did t he cells go for serum starvation? Nothing is 
mentioned in the manuscript regarding serum starvat ion before cells are going to be treated 
with niclosamide. It is needed to mention, because it is necessary to have serum starvation 
before going to treat the cells to get all cells at  the same cell cycle phase. 
5. It is needed to discuss clearly regarding the in hibitors and shRNA using in this study in the 
methods section. (It could be discussed in the sepa rate paragraph in the method section).  
6. Additional experiment is needed to confirm the a poptotic signaling (example: to see the 
DNA degradation of niclosamide treated cells and ce lls treated with DMSO by agarose gel 
electrophoresis) 
7. Figure and Legends need to rewrite. Better to fo llow any published article to follow the 
way the Figure and Legends should be written. 
8. In "Result" section under the first title "Niclo samide suppressed cells growth by inducing 
ER-stress in HCC cells", there is couple of sentenc es that need to be removed from this 
section to "Discussion section" (Such as, Current s tudies have demonstrated that ATF3 
plays an integral role … modulate cell apoptosis [9]). 
9. In the "Result" section there is another title t hat needs to be changed for an example, 
instead of this title "Cell apoptosis induced by ni closamide could be suppressed by 
inhibitor of the PERK pathway" the title should be "Niclosamide induced apoptosis in the 
HCC suppressed by PERK inhibition" 
10. And another title in the "Result" section needs  to be changed. Instead of this title "ATF3 
plays a central role in cell apoptosis induced by n iclosamide" the title should be "ATF3 
upregulation during cell apoptosis induced by niclo samide" 
11. Need to follow the manuscript format criteria f or this journal. Especially in the reference 
section. 
 
Are the methods appropriate and well described? 
If not, please specify what is required in your com ments 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 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: 
Needs some language corrections before being publis hed 
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:  16 Nov 2015 
 
Reviewer:  Syeda Afroze 
 
Reviewer's report: 
This manuscript entitled "Niclosamide induces cell apoptosis through upregulating ATF3 and 
activating PERK pathway in Hepatocellular carcinoma  cells" submitted by Weng  et al, the 
authors aimed to evaluate the effect of Niclosamide  on the Hepatocellular carcinoma cells. There 
are lots of issue to promote this hepatocellular ca rcinoma, such as HBV, HCV, Non-alcoholic 
fatty liver disease (NAFLD), Non-alcoholic steatohe patitis (NASH), Alcohol intake and 
Hemochromatosis as well. Hepatocellular carcinoma ( HCC) is one of the most devastating 
malignancy in human around the world. Above all the  prognosis of this disease is very much 
challenging. Even after the detection and taking ca re of it, the retrieval from this disease is very 
poor due to the lack of proper drugs (chemotherapeu tic) which has less or no side effect and the 
other reason gradually tumor cells tend to get chem o-resistance. That is why the survey for the 
new drugs for HCC treatment is necessary. Since Nic losamide is an antihelminthic drug which 
has been used to treat tapeworm infections also has  been shown anticancer effect in different 
tumor cell types as well as leukemia, colon cancer and glioma. Therefore in this study the 
authors assessed the anticancerous functional activ ity and mechanism of Niclosamide on two 
different HCC cell lines HepG2 and QGY7701. To expl ore the anti-HCC function and 
mechanism of Niclosamide in these two HCC cell line s, the authors performed several 
experiments such as, Cell viability assay, western blot, qPCR, Flow Cytometry and also did 
immunofluorescence in response to Niclosamide. The authors performed qPCR for evaluating 
these genes expressions such as, mRNA expression of  PERK, ATF3, ATF4 and CHOP, which 
are related to PERK signaling pathway. The authors also demonstrated western blot to see the 
phosphorylation of PERK (pPERK), protein expression  of caspase-3, PARP, PERK, CHOP, 
ATF3 and ATF4.The authors did Immunofluorescence st udy to see the location of expression of 
ATF3 and CHOP in these two HCC cell lines as well. The authors also conducted experiment by 
using PERK inhibitor GSK2606414 and knockdown the A TF3 gene to confirm that nicloamide 
is activating apoptosis through this PERK pathway v ia the upregulating ATF3 espression.It is an 
interesting study and work has done very well. But the presentation of this manuscript is not well 
organized. And also some certain aspects not well d escribed. So before going for final 
submission of this manuscript, it is recommended to  take care of this manuscript in terms of the 
following extensive major revision. Major concerns are mentioned below: 
 
Major Concerns: 
1. Title should be retitled. For an example, title could be "Niclosamide induced cell apoptosis 
via upregulation of ATF3 and activation of PERK in Hepatocellular carcinoma cells."   
2. The abstract and the manuscript should be carefu lly checked in terms of grammar. Need to 
revise by Scientific English editor. 
3. There are some information in the Background sec tion need to be removed from there and 
add it to the Materials and Methods section and res ult section as well. (Example: Last 
paragraph of the Background section "For investigat ing niclosamide anti-HCC function and 
mechanism, HepG2 and QGY7701 HCC cell lines…… , an inhibitor of PERK.) 
4. Before treating the cells with niclosamide did t he cells go for serum starvation? Nothing is 
mentioned in the manuscript regarding serum starvat ion before cells are going to be treated 
with niclosamide. It is needed to mention, because it is necessary to have serum starvation 
before going to treat the cells to get all cells at  the same cell cycle phase. 
5. It is needed to discuss clearly regarding the in hibitors and shRNA using in this study in the 
methods section. (It could be discussed in the sepa rate paragraph in the method section).  
6. Additional experiment is needed to confirm the a poptotic signaling (example: to see the 
DNA degradation of niclosamide treated cells and ce lls treated with DMSO by agarose gel 
electrophoresis) 
7. Figure and Legends need to rewrite. Better to fo llow any published article to follow the 
way the Figure and Legends should be written. 
8. In "Result" section under the first title "Niclo samide suppressed cells growth by inducing 
ER-stress in HCC cells", there is couple of sentenc es that need to be removed from this 
section to "Discussion section" (Such as, Current s tudies have demonstrated that ATF3 
plays an integral role … modulate cell apoptosis [9]). 
9. In the "Result" section there is another title t hat needs to be changed for an example, 
instead of this title "Cell apoptosis induced by ni closamide could be suppressed by 
inhibitor of the PERK pathway" the title should be "Niclosamide induced apoptosis in the 
HCC suppressed by PERK inhibition" 
10. And another title in the "Result" section needs  to be changed. Instead of this title "ATF3 
plays a central role in cell apoptosis induced by n iclosamide" the title should be "ATF3 
upregulation during cell apoptosis induced by niclo samide" 
11. Need to follow the manuscript format criteria f or this journal. Especially in the reference 
section. 
 
Are the methods appropriate and well described? 
If not, please specify what is required in your com ments 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 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: 
Needs some language corrections before being publis hed 
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:  16 Nov 2015 
 
Reviewer:  Syeda Afroze 
 
Reviewer's report: 
This manuscript entitled "Niclosamide induces cell apoptosis through upregulating ATF3 and 
activating PERK pathway in Hepatocellular carcinoma  cells" submitted by Weng  et al, the 
authors aimed to evaluate the effect of Niclosamide  on the Hepatocellular carcinoma cells. There 
are lots of issue to promote this hepatocellular ca rcinoma, such as HBV, HCV, Non-alcoholic 
fatty liver disease (NAFLD), Non-alcoholic steatohe patitis (NASH), Alcohol intake and 
Hemochromatosis as well. Hepatocellular carcinoma ( HCC) is one of the most devastating 
malignancy in human around the world. Above all the  prognosis of this disease is very much 
challenging. Even after the detection and taking ca re of it, the retrieval from this disease is very 
poor due to the lack of proper drugs (chemotherapeu tic) which has less or no side effect and the 
other reason gradually tumor cells tend to get chem o-resistance. That is why the survey for the 
new drugs for HCC treatment is necessary. Since Nic losamide is an antihelminthic drug which 
has been used to treat tapeworm infections also has  been shown anticancer effect in different 
tumor cell types as well as leukemia, colon cancer and glioma. Therefore in this study the 
authors assessed the anticancerous functional activ ity and mechanism of Niclosamide on two 
different HCC cell lines HepG2 and QGY7701. To expl ore the anti-HCC function and 
mechanism of Niclosamide in these two HCC cell line s, the authors performed several 
experiments such as, Cell viability assay, western blot, qPCR, Flow Cytometry and also did 
immunofluorescence in response to Niclosamide. The authors performed qPCR for evaluating 
these genes expressions such as, mRNA expression of  PERK, ATF3, ATF4 and CHOP, which 
are related to PERK signaling pathway. The authors also demonstrated western blot to see the 
phosphorylation of PERK (pPERK), protein expression  of caspase-3, PARP, PERK, CHOP, 
ATF3 and ATF4.The authors did Immunofluorescence st udy to see the location of expression of 
ATF3 and CHOP in these two HCC cell lines as well. The authors also conducted experiment by 
using PERK inhibitor GSK2606414 and knockdown the A TF3 gene to confirm that nicloamide 
is activating apoptosis through this PERK pathway v ia the upregulating ATF3 espression.It is an 
interesting study and work has done very well. But the presentation of this manuscript is not well 
organized. And also some certain aspects not well d escribed. So before going for final 
submission of this manuscript, it is recommended to  take care of this manuscript in terms of the 
following extensive major revision. Major concerns are mentioned below: 
 
Major Concerns: 
1. Title should be retitled. For an example, title could be "Niclosamide induced cell apoptosis 
via upregulation of ATF3 and activation of PERK in Hepatocellular carcinoma cells."   
2. The abstract and the manuscript should be carefu lly checked in terms of grammar. Need to 
revise by Scientific English editor. 
3. There are some information in the Background sec tion need to be removed from there and 
add it to the Materials and Methods section and res ult section as well. (Example: Last 
paragraph of the Background section "For investigat ing niclosamide anti-HCC function and 
mechanism, HepG2 and QGY7701 HCC cell lines…… , an inhibitor of PERK.) 
4. Before treating the cells with niclosamide did t he cells go for serum starvation? Nothing is 
mentioned in the manuscript regarding serum starvat ion before cells are going to be treated 
with niclosamide. It is needed to mention, because it is necessary to have serum starvation 
before going to treat the cells to get all cells at  the same cell cycle phase. 
5. It is needed to discuss clearly regarding the in hibitors and shRNA using in this study in the 
methods section. (It could be discussed in the sepa rate paragraph in the method section).  
6. Additional experiment is needed to confirm the a poptotic signaling (example: to see the 
DNA degradation of niclosamide treated cells and ce lls treated with DMSO by agarose gel 
electrophoresis) 
7. Figure and Legends need to rewrite. Better to fo llow any published article to follow the 
way the Figure and Legends should be written. 
8. In "Result" section under the first title "Niclo samide suppressed cells growth by inducing 
ER-stress in HCC cells", there is couple of sentenc es that need to be removed from this 
section to "Discussion section" (Such as, Current s tudies have demonstrated that ATF3 
plays an integral role … modulate cell apoptosis [9]). 
9. In the "Result" section there is another title t hat needs to be changed for an example, 
instead of this title "Cell apoptosis induced by ni closamide could be suppressed by 
inhibitor of the PERK pathway" the title should be "Niclosamide induced apoptosis in the 
HCC suppressed by PERK inhibition" 
10. And another title in the "Result" section needs  to be changed. Instead of this title "ATF3 
plays a central role in cell apoptosis induced by n iclosamide" the title should be "ATF3 
upregulation during cell apoptosis induced by niclo samide" 
11. Need to follow the manuscript format criteria f or this journal. Especially in the reference 
section. 
 
Are the methods appropriate and well described? 
If not, please specify what is required in your com ments 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 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: 
Needs some language corrections before being publis hed 
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