Title: A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation
of autophagy in human gastric cells.
Version: 1 Date: 22 May 2023
Reviewer's report:
This manuscript by Sakatani et al. describes the effect of H. pylori CagA protein on autophagy.
Role of CagA in autophagy has little interest until now, so data in this manuscript would be
interesting to the field. I think this manuscript is well organized, but following issued should
be adressed.
Methods
1. Western blotting
(1) Is phospho-tyrosine antibody used to detect CagA protein? If so, clarify it in method or result
section.
(2) Add information about antibodies for P62, ATG7, and ULK1 also.
2. qRT-PCR
(1) Describe PCR conditions
Results
1. line 170: For the better understanding of readers with various backgrounds, I think it should
be explained in the introduction what the 'autophagic flux' is.
2. Fig 1A: It is written like that autophagic flux index was used to measure the effect of H. pylori
infection (lines 170-172), but autophagic flux index cannot be calculated by the data shown in
fig 1A because chloroquine was not used in that experiment. It should be described that why the
autophagic flux was regarded to be decreased by H. pylori infection in Fig 1A.
3. Fig 1: This figure contains many sub-panels (e.g. left and right for Fig 1A. left/middle/right
and upper/lower for Fig 1B). It would be better if each sub-panels are clearly indicated in the
main text. This applies to all figures in this manuscript.
4. line 173: It looks like that the terms 'autophagic flux index' and 'autophagic index' are both
used in the manuscript. Unify this.
5. Fig 1B: P62 also is an important marker of autophagy. It would be better if a description about
P62 data is provided.
6. Fig 2: Describe timeframe of the experiment, e.g. how long the expression was induced etc.
7. Fig 2A: Fig 2A shows the result of DNA gel electrophoresis, not "autophagic index of RGM1-
Mock and RGM1-CagA cells" (lines 180-181). Cite the figure in the proper place. In addition,
it would be clearer if CagA protein expression data can be shown.
8. lines 181-182: What is being suppressed is 'autophagy', not 'autophagic index'. Autophagic
index is an abstract value, so it can be increased or decreased, but cannot be suppressed or
activated. Please check this throughout the manuscript.
9. Fig 3A: It would be better if explanation of ATG5, ATG7, and ULK1 data is added in the
main text.
10. Fig 4F: Add vertical axis title in the bar chart of Fig 4F.
11. Figure legends: Usually, figure legends do not contain the interpretation of data, except the
legend title. I recommend to move such interpretations to the main text, and leave the description
about the experiment in the legends.
Discussion
1. line 223: No proinflammatory cytokine was measured in this study. Generally, COX-2 is not
classified as a proinflammatory 'cytokine'. Rephrase the sentense. The same applies to lines 33,
48, and 97.
2. line 275: I think that 'BECN1 mRNA expression was decreased after H.pylori infection' was
not demonstrated in this study. Fig 3A shows Beclin1 protein expression. Fig 3B shows Beclin1
mRNA and suggests that Hp-WT induces less Beclin1 transcription than Hp-dcagA but it do not
show that 'BECN1 mRNA expression was decreased after H.pylori infection' because it lacks
control.
Title: A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation
of autophagy in human gastric cells.
Version: 1 Date: 22 May 2023
Reviewer's report:
This manuscript by Sakatani et al. describes the effect of H. pylori CagA protein on autophagy.
Role of CagA in autophagy has little interest until now, so data in this manuscript would be
interesting to the field. I think this manuscript is well organized, but following issued should
be adressed.
Methods
1. Western blotting
(1) Is phospho-tyrosine antibody used to detect CagA protein? If so, clarify it in method or result
section.
(2) Add information about antibodies for P62, ATG7, and ULK1 also.
2. qRT-PCR
(1) Describe PCR conditions
Results
1. line 170: For the better understanding of readers with various backgrounds, I think it should
be explained in the introduction what the 'autophagic flux' is.
2. Fig 1A: It is written like that autophagic flux index was used to measure the effect of H. pylori
infection (lines 170-172), but autophagic flux index cannot be calculated by the data shown in
fig 1A because chloroquine was not used in that experiment. It should be described that why the
autophagic flux was regarded to be decreased by H. pylori infection in Fig 1A.
3. Fig 1: This figure contains many sub-panels (e.g. left and right for Fig 1A. left/middle/right
and upper/lower for Fig 1B). It would be better if each sub-panels are clearly indicated in the
main text. This applies to all figures in this manuscript.
4. line 173: It looks like that the terms 'autophagic flux index' and 'autophagic index' are both
used in the manuscript. Unify this.
5. Fig 1B: P62 also is an important marker of autophagy. It would be better if a description about
P62 data is provided.
6. Fig 2: Describe timeframe of the experiment, e.g. how long the expression was induced etc.
7. Fig 2A: Fig 2A shows the result of DNA gel electrophoresis, not "autophagic index of RGM1-
Mock and RGM1-CagA cells" (lines 180-181). Cite the figure in the proper place. In addition,
it would be clearer if CagA protein expression data can be shown.
8. lines 181-182: What is being suppressed is 'autophagy', not 'autophagic index'. Autophagic
index is an abstract value, so it can be increased or decreased, but cannot be suppressed or
activated. Please check this throughout the manuscript.
9. Fig 3A: It would be better if explanation of ATG5, ATG7, and ULK1 data is added in the
main text.
10. Fig 4F: Add vertical axis title in the bar chart of Fig 4F.
11. Figure legends: Usually, figure legends do not contain the interpretation of data, except the
legend title. I recommend to move such interpretations to the main text, and leave the description
about the experiment in the legends.
Discussion
1. line 223: No proinflammatory cytokine was measured in this study. Generally, COX-2 is not
classified as a proinflammatory 'cytokine'. Rephrase the sentense. The same applies to lines 33,
48, and 97.
2. line 275: I think that 'BECN1 mRNA expression was decreased after H.pylori infection' was
not demonstrated in this study. Fig 3A shows Beclin1 protein expression. Fig 3B shows Beclin1
mRNA and suggests that Hp-WT induces less Beclin1 transcription than Hp-dcagA but it do not
show that 'BECN1 mRNA expression was decreased after H.pylori infection' because it lacks
control.
Please review this paper.
user
Title: A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation
of autophagy in human gastric cells.
Version: 1 Date: 22 May 2023
Reviewer's report:
This manuscript by Sakatani et al. describes the effect of H. pylori CagA protein on autophagy.
Role of CagA in autophagy has little interest until now, so data in this manuscript would be
interesting to the field. I think this manuscript is well organized, but following issued should
be adressed.
Methods
1. Western blotting
(1) Is phospho-tyrosine antibody used to detect CagA protein? If so, clarify it in method or result
section.
(2) Add information about antibodies for P62, ATG7, and ULK1 also.
2. qRT-PCR
(1) Describe PCR conditions
Results
1. line 170: For the better understanding of readers with various backgrounds, I think it should
be explained in the introduction what the 'autophagic flux' is.
2. Fig 1A: It is written like that autophagic flux index was used to measure the effect of H. pylori
infection (lines 170-172), but autophagic flux index cannot be calculated by the data shown in
fig 1A because chloroquine was not used in that experiment. It should be described that why the
autophagic flux was regarded to be decreased by H. pylori infection in Fig 1A.
3. Fig 1: This figure contains many sub-panels (e.g. left and right for Fig 1A. left/middle/right
and upper/lower for Fig 1B). It would be better if each sub-panels are clearly indicated in the
main text. This applies to all figures in this manuscript.
4. line 173: It looks like that the terms 'autophagic flux index' and 'autophagic index' are both
used in the manuscript. Unify this.
5. Fig 1B: P62 also is an important marker of autophagy. It would be better if a description about
P62 data is provided.
6. Fig 2: Describe timeframe of the experiment, e.g. how long the expression was induced etc.
7. Fig 2A: Fig 2A shows the result of DNA gel electrophoresis, not "autophagic index of RGM1-
Mock and RGM1-CagA cells" (lines 180-181). Cite the figure in the proper place. In addition,
it would be clearer if CagA protein expression data can be shown.
8. lines 181-182: What is being suppressed is 'autophagy', not 'autophagic index'. Autophagic
index is an abstract value, so it can be increased or decreased, but cannot be suppressed or
activated. Please check this throughout the manuscript.
9. Fig 3A: It would be better if explanation of ATG5, ATG7, and ULK1 data is added in the
main text.
10. Fig 4F: Add vertical axis title in the bar chart of Fig 4F.
11. Figure legends: Usually, figure legends do not contain the interpretation of data, except the
legend title. I recommend to move such interpretations to the main text, and leave the description
about the experiment in the legends.
Discussion
1. line 223: No proinflammatory cytokine was measured in this study. Generally, COX-2 is not
classified as a proinflammatory 'cytokine'. Rephrase the sentense. The same applies to lines 33,
48, and 97.
2. line 275: I think that 'BECN1 mRNA expression was decreased after H.pylori infection' was
not demonstrated in this study. Fig 3A shows Beclin1 protein expression. Fig 3B shows Beclin1
mRNA and suggests that Hp-WT induces less Beclin1 transcription than Hp-dcagA but it do not
show that 'BECN1 mRNA expression was decreased after H.pylori infection' because it lacks
control.
assistant