Reviewer ’s report  
 
Title: Cobalt Chloride Compromises Transepithelial Barrier Properties of CaCo -2 BBe Human 
Gastrointestinal Epithelial Cell Layers  
 
Version: 0 Date: 30 Mar 2017  
 
Reviewer: David  Lo 
 
Reviewer's report:  
 
The paper presents data on Caco -2 stu dies in the presence of inhibitors of HIF -1 degradation. 
The authors conclude that cobalt ions and other inhibitors of HIF -1 degradation can directly 
result in decreases in Caco -2 monolayer permeability.  
 
 
The data is not clearly presented and much of the e xpected primary data such as micrographs of 
monolayer integrity, Western blots, and so on, are not presented or only shown as bar graphs. 
The figures are poorly labeled if at all (e.g., Figure 1 is not well labeled, and most could be 
combined into a smalle r number of data figures.  
 
 
On the basic data itself, there is a reliance on an effect seen mainly in rather immature (7 day) 
monolayers not seen in mature monolayers (we have no images of the tight junctions to help 
assess monolayer maturity). Thus its re levance to in vivo biology is quite uncertain. To 
compound these concerns, the data on tight junction protein levels was apparently generated 
from monolayer cultures in flasks and not in transwell filters, so again its applicability to the 
transwell data i s not clear.  
 
 
The inhibition of HIF -1 degradation is supposed to be the focus of the study, but no data are 
provided to show the levels of HIF -1 in cells except in the case of the knockdown cell lines. The 
assumed effect on HIF -1 is also apparently studied  in the absence of any stressors that would 
potentially reveal parallel mechanisms affecting tight junction function, or increases in HIF -1 
transcript or protein levels. In addition, there is no analysis of effects on gene targets of HIF -1 
action in the nu cleus.  
 
 
So in the end, the data have a number of problems, including concerns over how it would be of 
relevance to more physiological in vivo situations, and there is a lot of missing information that 
would be of specific relevance to tight junction and HIF -1 acti vity. 
 
 
 
 
 
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.  
 
No 
 
Are the conclusions drawn adequatel y 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 wo uld you recommend an 
additional statistical review?  
If an additional statistical review is recommend ed, please specify what aspects require further 
assessment in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Are the methods appropriate and well described?  
If not, please specify what is required i n your comments to the authors.  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way ga in 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 co ntent 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 inter ests' 
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 t hat my name will be included 
on my report to the authors and, if the m anuscript is accepted f or 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 und er an Open Access  Creative Commons 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that a ny 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 t he journal  
 
Reviewer ’s report  
 
Title: Cobalt Chloride Compromises Transepithelial Barrier Properties of CaCo -2 BBe Human 
Gastrointestinal Epithelial Cell Layers  
 
Version: 0 Date: 30 Mar 2017  
 
Reviewer: David  Lo 
 
Reviewer's report:  
 
The paper presents data on Caco -2 stu dies in the presence of inhibitors of HIF -1 degradation. 
The authors conclude that cobalt ions and other inhibitors of HIF -1 degradation can directly 
result in decreases in Caco -2 monolayer permeability.  
 
 
The data is not clearly presented and much of the e xpected primary data such as micrographs of 
monolayer integrity, Western blots, and so on, are not presented or only shown as bar graphs. 
The figures are poorly labeled if at all (e.g., Figure 1 is not well labeled, and most could be 
combined into a smalle r number of data figures.  
 
 
On the basic data itself, there is a reliance on an effect seen mainly in rather immature (7 day) 
monolayers not seen in mature monolayers (we have no images of the tight junctions to help 
assess monolayer maturity). Thus its re levance to in vivo biology is quite uncertain. To 
compound these concerns, the data on tight junction protein levels was apparently generated 
from monolayer cultures in flasks and not in transwell filters, so again its applicability to the 
transwell data i s not clear.  
 
 
The inhibition of HIF -1 degradation is supposed to be the focus of the study, but no data are 
provided to show the levels of HIF -1 in cells except in the case of the knockdown cell lines. The 
assumed effect on HIF -1 is also apparently studied  in the absence of any stressors that would 
potentially reveal parallel mechanisms affecting tight junction function, or increases in HIF -1 
transcript or protein levels. In addition, there is no analysis of effects on gene targets of HIF -1 
action in the nu cleus.  
 
 
So in the end, the data have a number of problems, including concerns over how it would be of 
relevance to more physiological in vivo situations, and there is a lot of missing information that 
would be of specific relevance to tight junction and HIF -1 acti vity. 
 
 
 
 
 
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.  
 
No 
 
Are the conclusions drawn adequatel y 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 wo uld you recommend an 
additional statistical review?  
If an additional statistical review is recommend ed, please specify what aspects require further 
assessment in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Are the methods appropriate and well described?  
If not, please specify what is required i n your comments to the authors.  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way ga in 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 co ntent 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 inter ests' 
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 t hat my name will be included 
on my report to the authors and, if the m anuscript is accepted f or 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 und er an Open Access  Creative Commons 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that a ny 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 t he journal  
 
Please review this paper.
user
Reviewer ’s report  
 
Title: Cobalt Chloride Compromises Transepithelial Barrier Properties of CaCo -2 BBe Human 
Gastrointestinal Epithelial Cell Layers  
 
Version: 0 Date: 30 Mar 2017  
 
Reviewer: David  Lo 
 
Reviewer's report:  
 
The paper presents data on Caco -2 stu dies in the presence of inhibitors of HIF -1 degradation. 
The authors conclude that cobalt ions and other inhibitors of HIF -1 degradation can directly 
result in decreases in Caco -2 monolayer permeability.  
 
 
The data is not clearly presented and much of the e xpected primary data such as micrographs of 
monolayer integrity, Western blots, and so on, are not presented or only shown as bar graphs. 
The figures are poorly labeled if at all (e.g., Figure 1 is not well labeled, and most could be 
combined into a smalle r number of data figures.  
 
 
On the basic data itself, there is a reliance on an effect seen mainly in rather immature (7 day) 
monolayers not seen in mature monolayers (we have no images of the tight junctions to help 
assess monolayer maturity). Thus its re levance to in vivo biology is quite uncertain. To 
compound these concerns, the data on tight junction protein levels was apparently generated 
from monolayer cultures in flasks and not in transwell filters, so again its applicability to the 
transwell data i s not clear.  
 
 
The inhibition of HIF -1 degradation is supposed to be the focus of the study, but no data are 
provided to show the levels of HIF -1 in cells except in the case of the knockdown cell lines. The 
assumed effect on HIF -1 is also apparently studied  in the absence of any stressors that would 
potentially reveal parallel mechanisms affecting tight junction function, or increases in HIF -1 
transcript or protein levels. In addition, there is no analysis of effects on gene targets of HIF -1 
action in the nu cleus.  
 
 
So in the end, the data have a number of problems, including concerns over how it would be of 
relevance to more physiological in vivo situations, and there is a lot of missing information that 
would be of specific relevance to tight junction and HIF -1 acti vity. 
 
 
 
 
 
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.  
 
No 
 
Are the conclusions drawn adequatel y 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 wo uld you recommend an 
additional statistical review?  
If an additional statistical review is recommend ed, please specify what aspects require further 
assessment in your comments to the editors.  
 
I recommend additional statistical review  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Are the methods appropriate and well described?  
If not, please specify what is required i n your comments to the authors.  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way ga in 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 co ntent 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 inter ests' 
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 t hat my name will be included 
on my report to the authors and, if the m anuscript is accepted f or 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 und er an Open Access  Creative Commons 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that a ny 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 t he journal  
 
assistant