Recombinant Type XVII Collagen Promotes Hair Growth by Activating the Wnt/β-Catenin and SHH/GLI Signaling Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Your results show that rhCOL17a activates Wnt and Shh signalling in human dermal papilla fibroblasts and provides protection against UVB-induced apoptosis. Along this line, application of rhCOL17 promotes hair growth in mice along with activation of Wnt and SHH pathway. Overall those data are interesting, however I am concern about some methods used in your study and on the link between the two models used to draw the conclusions, in particular:

1. It is clear from the representative images of COL17 in mouse that its expression is not concentrated to the dermal papilla but rather, as shown before, around the bulge stem cells. Thus, what is the rationale to use dermal papilla cells?
2. To my knowledge COL17 is produced by keratinocytes, how can you explain its expression in dermal papilla cells in absence of epithelial cells?
3. I do not also understand the rationale for using UVB challenge in the dermal papilla fibroblasts. Usually dermal papilla fibroblasts do not undergo apoptosis during anagen catagen transition, but stop to produce morphogens (incl those you have evaluated, i.e. VEGF, FGF-7) and migrate out in the dermal sheath. Thus, the rational to use UVB for me is not clear. Why testosterone was not use to align with the mouse model?
4. It is well known that dermal papilla fibroblasts in 2D rapidly loose their phenotype. Thus, I encourage the authors to perform additional experiment with dermal papilla cells in 3D with testosterone to align with the data in the mouse model.

Minor points:

-Abstract: It is missing in the background as rationale the findings of the the two major papers on COL17 role in the hair follicle

-Abstract: the models are not clearly described

-Abstract: in the results you refer to stam cells rather than dermal papilla fibroblasts and it is not clear what is new

-there are some mistakes in terminology here and there: e.g. hair follicle atrophy is a term that is not used, line 53 it should be telogen rather than catagen

-results: indicate recombinant COL17 as rhCOL17 and endogenous COL17 as endCOL17, this would help the reader understaning the difference.

-Results: subsection title is not informative for the findings

-Pictures: not sure whether it the pdf, but the quality is very low

-Discussion: this is not a discussion but a summary of the results, it requires extensive work

 

Comments on the Quality of English Language

The english is overall very poor, please check e.g. line 80-81, 99-100, 229-230.

Author Response

Comments 1: [I do not also understand the rationale for using UVB challenge in the dermal papilla fibroblasts. Usually dermal papilla fibroblasts do not undergo apoptosis during anagen catagen transition, but stop to produce morphogens (incl those you have evaluated, i.e. VEGF, FGF-7) and migrate out in the dermal sheath. Thus, the rational to use UVB for me is not clear. It is clear from the representative images of COL17 in mouse that its expression is not concentrated to the dermal papilla but rather, as shown before, around the bulge stem cells. Thus, what is the rationale to use dermal papilla cells?]

Responses 1: [We appreciate this thoughtful question. Both hair follicle stem cells (HFSCs) and Hair Follicle dermal papilla cells (HFDPCs) play critical roles in hair regeneration. However, given the technical challenges in isolating HFSCs and the inherent difficulty in maintaining their stemness in vitro, we selected HFDPCs as the primary cellular model for this investigation.HFDPC is a key mesenchymal cell in hair follicles and is widely used in the research of hair growth regulation, hair loss mechanisms and drug development. HFDPC is located in the dermal papilla region and directly participates in the initiation and maintenance of the hair follicle cycle (anagen phase, catagen phase, resting phase) by secreting growth factors (such as VEGF, KGF, IL-8, etc.) and regulating signaling pathways (such as SHH, Wnt). HFDPC has been used in multiple studies to elucidate the molecular mechanism of androgenetic alopecia (AGA).]

 

 

Comments 2: [To my knowledge COL17 is produced by keratinocytes, how can you explain its expression in dermal papilla cells in absence of epithelial cells?]

Response 2 [It is currently believed that COL17A1 is mainly expressed by keratinocytes (epithelial cells) in the basal layer, serving as a key component of hemidesmosomes to anchor the epidermis to the dermis. As we know that type XVII collagen affects the growth of hair follicle stem cells, we therefore aimed to detect whether type XVII collagen is present in HFDPCs. The results from cell climbing slides showed that rhCOL17A1 increased the expression of type XVII collagen in HFDPC cells.]

Comments 3:[Why testosterone was not use to align with the mouse model?]

Response 3[At the cellular level, we mainly detected growth factors, and growth factors are related to the apoptosis of hair follicle cells. Therefore, we detected Bax and Bcl-2.UVB can rapidly induce degenerative changes in hair follicles (such as inhibiting VEGF secretion within 48 hours) and is suitable for the study of acute injury mechanisms. Here we would like to illustrate that ColXVII is beneficial for providing a favorable growth environment for hair follicles and promoting hair growth.]

 

Comments 3: [It is well known that dermal papilla fibroblasts in 2D rapidly loose their phenotype. Thus, I encourage the authors to perform additional experiment with dermal papilla cells in 3D with testosterone to align with the data in the mouse model.]

Response 3: [Thank you for your suggestion, reviewer. The 3D model of HFDPC cells is rather difficult to obtain. We are still continuing to study the effect of COL17A1 on hair loss. In the subsequent research, we will consider adopting your suggestion, but this will require a relatively long trial period.]

 

Comments 4: [Abstract: It is missing in the background as rationale the findings of the the two major papers on COL17 role in the hair follicle.]

Response 4: [We have added the research results of these two key papers in the background section to enrich the research background.]

 

Comments 5: [ Abstract-In the results you refer to stam cells rather than dermal papilla fibroblasts and it is not clear what is new.]

 

Response 5: [Thank you for pointing this out; it was my mistake. The correct term should be hair follicle dermal papilla cells (HFDPCs), and I've updated it in the manuscript.]

 

Comments 6: [results: indicate recombinant COL17 as rhCOL17 and endogenous COL17 as endCOL17, this would help the reader understaning the difference.]

Response 6: [We changed CoL17 to rhCOL17A1，because it is derived from the α1 chain. The endogenous CoL17 was consistent with the expression in other studies and was COLXVII. The two have significant differences in form and are easier to distinguish.]

 

Comments 7: [Results: subsection title is not informative for the findings]

Response 7: [We modified the subsection title to make it more clearly indicate the research results of each part.]

Comments 8: Pictures: not sure whether it the pdf, but the quality is very low

Response 8: [We have re-adjusted the image format to TIFF and ensured the resolution is above 600 dpi.]

Comments 9: [Discussion: this is not a discussion but a summary of the results, it requires extensive work.]

Response 9: [In the results section, we have added discussions on the current research status and the future application prospects of rhCOL17A1.]

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the role of humanized recombinant type XVII collagen （Col17A1） in preventing hair loss, demonstrating significant application value.  Col17A1 regulates hair growth-related signaling pathways in HFDPCs (hair follicle dermal papilla cells), thereby exerting anti-hair loss effects.  Recombinant humanized collagen shows superior safety compared to conventional hair loss prevention ingredients and holds promise for future applications in addressing androgenetic alopecia and follicular subhealth conditions.  Regarding this research paper, I offer the following suggestions for improvement:

1. The sentence structures in the Results section of the abstract are somewhat monotonous. It is recommended to revise and polish them to enhance diversity.
2. Pay attention to the usage of the "be verb“ in the paper. For example, the usage of "was" in line 137 is incorrect.
3. Pay attention to the use of articles in the paper. For example, the article "a" in line 139 should be deleted.
4. The writing in academic papers should be as concise and clear as possible, avoiding overly complex sentences.
5. Modify the format of punctuation marks to make them uniform.
6. All figure legends should indicate the sample size for each experimental group.
7. The magnification must be indicated for the HE-stained sections in Figure 4.
8. For the q-PCR and Western blot (WB) experimental results presented in bar graphs, provide specific numerical values with SD (standard deviation) values for verification. These could be submitted as supplementary materials.

It is recommended to expand on the description of future application prospects.

Author Response

Thank you very much for your valuable suggestions. The following is my reply.

Comments 1: [The sentence structures in the Results section of the abstract are somewhat monotonous. It is recommended to revise and polish them to enhance diversity.]

Response 1:[The sentence structures were modified to enhance linguistic diversity and avoid repetitive phrasing in the translated text.]

 

Comments 2: [Pay attention to the usage of the "be verb” in the paper. For example, the usage of "was" in line 137 is incorrect.]

Response 2: [The verb form 'was' has been replaced with 'were' in the relevant context.]

 

Comments 3:[Pay attention to the use of articles in the paper. For example, the article "a" in line 139 should be deleted]

Response 3: [We have addressed this error in the updated manuscript.]

 

Comments 4: [The writing in academic papers should be as concise and clear as possible, avoiding overly complex sentences.]

Response 4: [We have streamlined convoluted sentence structures in the draft to improve readability.]

 

Comments 5:[Modify the format of punctuation marks to make them uniform.]

Response 5: [We have harmonized all punctuation marks in the manuscript to ensure stylistic consistency.]

 

Comments 6: [All figure legends should indicate the sample size for each experimental group.]

Response 6:[We have marked the sample sizes of each experiment in the manuscript.]

 

Comment 7: [The magnification must be indicated for the HE-stained sections in Figure 4.]

Response 7:[We have added scale bars to all the magnified pictures.]

 

 

Comments 8:[For the q-PCR and Western blot (WB) experimental results presented in bar graphs, provide specific numerical values with SD (standard deviation) values for verification. These could be submitted as supplementary materials.]

Response 8:[For the q-PCR and Western blot (WB) experimental results, We have compiled the Mean and SD values for all metrics and uploaded them as supplementary material.]

 

Comments 9: [It is recommended to expand on the description of future application prospects.]

Response 9: [Thank you for your suggestion. We have added content regarding the potential applications of COL17A1 in hair care products to the Discussion section.]

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript clearly demonstrates the hair-promoting efficacy by recombination of type XVII collagen and thoroughly describes its mechanism of action through both the activation of the Wnt/β-catenin and SHH/GLI pathway and the inhibition of the MMP-9.  I have the following questions for the authors to address and provide revision suggestions for consideration:

1. Could the authors elaborate on the rationale for the concentration selection of Col17A1 in the cellular experiments?
2. Does the Col17A1 used in this study represent the full-length human type XVII collagen protein or a partial sequence? It is recommended to include the recombinant protein sequence in the supplementary materials.
3. The introduction section provides insufficient discussion on the current research status of hair loss ingredients. It is recommended to expand this part.
4. The results section lacks precision in describing the magnitude of increases/decreases. Providing quantitative proportions (e.g., fold-changes or percentages) is advised. To balance conciseness and clarity, the selective and precise presentation of research findings is appropriate.
5. The Discussion section lacks an overview of the current research status. The authors are advised to elaborate on the existing research to highlight the necessity of this study.

Author Response

We thank the reviewer for their insightful comments and valuable suggestions. These suggestions have helped us significantly improve the quality of our manuscript. 

Comments 1: [Could the authors elaborate on the rationale for the concentration selection of Col17A1 in the cellular experiments?]

Response 1:[Prior to the formal experiments, we conducted preliminary gradient concentration tests (e.g., 0.1–4 mg/mL) to screen appropriate drug concentrations based on CCK-8 assay results. The experimental data demonstrated that concentrations of 0.25, 0.5, and 1 mg/mL not only preserved cell viability but also exhibited enhancement effect on cellular activity.]

 

Comments 2: [Does the Col17A1 used in this study represent the full-length human type XVII collagen protein or a partial sequence? It is recommended to include the recombinant protein sequence in the supplementary materials.]

Response 2: [Col17A1 is not the full-length type XVII collagen but a truncated sequence derived from a specific region of the human type XVII collagen. The detailed amino acid sequence has been provided as Supplementary Material.]

 

Comments 3: [The introduction section provides insufficient discussion on the current research status of hair loss ingredients. It is recommended to expand this part.]

Response 3: [The Introduction section has been revised to expand on recent advances in plant extract-based therapies for alopecia, incorporating additional analyses of their molecular mechanisms.  This enhancement provides a more comprehensive foundation for contextualizing the study's objectives.]

 

Comments 4: [The results section lacks precision in describing the magnitude of increases/decreases. Providing quantitative proportions (e.g., fold-changes or percentages) is advised. To balance conciseness and clarity, the selective and precise presentation of research findings is appropriate.]

Response 4:[We provided a detailed description of the results and added the increase/decrease ratios of some experimental results to make the results more precise.]

 

Comments 5:[The Discussion section lacks an overview of the current research status. The authors are advised to elaborate on the existing research to highlight the necessity of this study.]

Response 5:[In the Discussion section, we have added an overview of the current raw material landscape for anti-hair loss hair care products, highlighting the advantages of recombinant collagen as an ingredient. We also describe the application potential of collagen in hair care formulations, making the manuscript more aligned with the journal's scope.]

 

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript explored the role of COL17A1 in hair regeneration and demonstrated a certain degree of innovation. However, the expression in the manuscript needs to be revised. The writing of some gene names and protein names does not comply with the standards, and some figures need to be modified. The specific comments are as follows:

 

1. The expression of the molecule "Col17A1" in the article is not standardized. Please revise it to the correct format (COL17A1 or Col17a1). For reference, see the expressions in the following two articles: "Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis" and "METTL14-mediated N6-methyladenosine modification of Col17a1/ Itgalpha6/ Itgbeta4 governs epidermal homeostasis".

 

2. In the Methods section of the abstract, "haematoxylin and eosin (HE)" should be revised to the standardized abbreviation format, "haematoxylin and eosin (H&E)".

 

3. In the Results section of the abstract, the expression “Col17A1 can increase endogenous ColXVII expression.” is neither clear nor scientific. Moreover, the abbreviation ColXVII has not been introduced previously in the text and should be defined when it first appears.

 

4. The abstract should preferably include a complete four-part structure: background, methods, results, and conclusions. Please add the conclusions section.

 

5. Merge these two paragraphs in the Introduction into one complete paragraph.

“ColXVII is a transmembrane protein that is primarily found in the basement membrane zone and is essential for the maintenance of skin structure and function.

ColXVII plays a crucial role in maintaining the structural integrity of hair follicles and establishing an optimal environment for hair growth 18, 19. ColXVII can interact with hair follicle stem cells and thus regulate their proliferation and differentiation. Moreover, ColXVII is essential for establishing the appropriate microenvironment that allows hair follicle stem cells to remain active and initiate the hair growth cycle when necessary.Furthermore, ColXVII may participate in various phases of the hair growth cycle......”

 

6. The last paragraph of the Introduction is a summary of the article's content. Please expand it, as it is currently too brief.

 

7. Please specify the age in weeks, gender, and hair follicle cycle stage of the mice used in the animal experiments, and supplement this information in the Methods section.

 

8. In the Fig2A, no significant differences were observed between the control and experimental groups for β-catenin and p-β-catenin. Please verify the data. The capitalization of β-actin should be consistent in the Fig2A and Fig2G.

 

9. Please add scale bars to the IF staining results, such as in the Fig3A, 4C, 5A, 5C, and 5E. When arranging the figures, maintain a consistent orientation of the hair follicles, such as from the upper right to the lower left.

 

10. In the Fig4E, only one representative image is needed for each experimental group, with the sampling days indicated. The orientation of the hair follicles should be consistent.

 

11. Please correct the format of gene and protein names throughout the manuscript.

Author Response

Comments 1: [The expression of the molecule "Col17A1" in the article is not standardized. Please revise it to the correct format (COL17A1 or Col17a1). For reference, see the expressions in the following two articles: "Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis" and "METTL14-mediated N6-methyladenosine modification of Col17a1/ Itgalpha6/ Itgbeta4 governs epidermal homeostasis]

Response 1: [We have fully accepted your suggestion, and to differentiate between recombinant type XVII collagen and endogenous type XVII collagen, the recombinant type XVII collagen will be designated as rhCOL17A1, while the endogenous type XVII collagen will be abbreviated as COLXVII.]

Comments 2: [In the Methods section of the abstract, "haematoxylin and eosin (HE)" should be revised to the standardized abbreviation format, "haematoxylin and eosin (H&E)"]

Response 2: [The "HE" has been changed to the correct format "H&E" in the full text.]

 

Comments 3: [In the Results section of the abstract, the expression “Col17A1 can increase endogenous ColXVII expression.” is neither clear nor scientific. Moreover, the abbreviation ColXVII has not been introduced previously in the text and should be defined when it first appears.]。

Response 3: [We have standardized the expression of COL17A1, with the abbreviation COLXVII specified as standing for type XVII collagen upon its first mention.]

Comments 4：[The abstract should preferably include a complete four-part structure: background, methods, results, and conclusions. Please add the conclusions section.Merge these two paragraphs in the Introduction into one complete paragraph]

Response 4: [We have added the conclusion section in the abstract.]

 Comments 5: [The last paragraph of the Introduction is a summary of the article's content. Please expand it, as it is currently too brief.]

Response 5:[We have expanded the concluding paragraph of the Introduction, describing the overall work to be undertaken in the paper.]

Comments 6: [Please specify the age in weeks, gender, and hair follicle cycle stage of the mice used in the animal experiments, and supplement this information in the Methods section.]

Response 6: [The age in weeks and gender of mice have been specified in the Materials section. As the mice exhibited asynchronous hair follicle cycles, we administered testosterone propionate to induce synchronized telogen phase, followed by topical application of  recombinant COL17A1 for interventional observation.]

 

Comments 7: [In the Fig2A, no significant differences were observed between the control and experimental groups for β-catenin and p-β-catenin. Please verify the data. The capitalization of β-actin should be consistent in the Fig2A and Fig2G.]

Response 7: [For the Western blot (WB) results, we performed normalization using ImageJ. While no discernible trend may be visually apparent, actual numerical differences were detected in the measurements. Furthermore, the final results are presented as the ratio of p-β-catenin to β-catenin (p-β-catenin/β-catenin), and this ratio format inherently amplifies differences between experimental groups. The format of β-actin in Figures 2a and 2g has been made consistent.]

Comments 8: [Please add scale bars to the IF staining results, such as in the Fig3A, 4C, 5A, 5C, and 5E. When arranging the figures, maintain a consistent orientation of the hair follicles, such as from the upper right to the lower left.]

Response 8: [We have now added scale bars to all immunofluorescence (IF) images. Regarding hair follicle orientation, the direction of paraffin-embedded skin tissue samples is inherently random during processing. Consequently, hair follicle alignment cannot be standardized in our final histological sections.]

 

Comments 9: [In the Fig4E, only one representative image is needed for each experimental group, with the sampling days indicated. The orientation of the hair follicles should be consistent.]

Response 9: [In Figure 4e, a representative image was placed for each experimental group. The sampling days are described in the methods and results sections of the article. Mice were euthanized on day 18, followed by H&E staining. Similar to our immunofluorescence (IF) results, consistent orientation of hair follicles could not be achieved in these histological sections.]

 

Comments 10: [Please correct the format of gene and protein names throughout the manuscript.]

Response 10: [We have corrected the names of the genes and proteins.]

 

Reviewer 5 Report

Comments and Suggestions for Authors

Dear Authors

Generally, this manuscript was well-organized relatively. 

However, there were many errors for improving your manuscript. 

Here were major comments. 

1. This manuscript has a basic problem, especially, their Western blot image.

Their gel image which they provided were not independent experiments with n number 3. The authors should provided at least 3 numbers of gel image in each protein data. 

2.ColXVII protein has been well-known for improving hair growth. Thus, the authors describe how to make or construct ColXVII protein in detail although this commercial purchase.  The authors also should add the related references about colXVII.

3. This manuscript has a small number of references and add more at least 50 references. 

4. This manuscript did not follow the policy of Cosmetics including the expression of abstract and reference. Please, check it. 

5. In writing the background in each result session, the authors did not include any reference and add the appropriate reference in each sentence, which derived from previous results. 

6. Please, add the scale bar in Figure 3, 4C/4E, 5, and 6 including the figure legend. 

 

Author Response

We thank the reviewer for their insightful comments and valuable suggestions. These suggestions have helped us significantly improve the quality of our manuscript. 

Comments 1:[This manuscript has a basic problem, especially, their Western blot image.Their gel image which they provided were not independent experiments with n number 3. The authors should provided at least 3 numbers of gel image in each protein data. ]

Respose 1: [We sorted out three gel images and uploaded them.]

Comments 2: [ColXVII protein has been well-known for improving hair growth. Thus, the authors describe how to make or construct ColXVII protein in detail although this commercial purchase.  The authors also should add the related references about colXVII.]

Respose 2: [The expression and purification of the recombinant collagen were accomplished by other researchers, and this study does not involve such experimental work. Therefore, we have only outlined its construction process concisely in the Methods section based on the patent (CN 118373900) documentation. ]

Comments 3: [This manuscript has a small number of references and add more at least 50 references. ]

Respose 3: [We enriched the results and discussion sections and added some references.]

Comments 4: [This manuscript did not follow the policy of Cosmetics including the expression of abstract and reference. Please, check it. ]

Respose 4: [We have added the results section to the abstract and modified its format to comply with Cosmetics journal requirements. Regarding references, the author guidelines indicate that specific formatting is not strictly required.]

Comments 5: [In writing the background in each result session, the authors did not include any reference and add the appropriate reference in each sentence, which derived from previous results. ]

Respose 5: [In the background in each result session, we cited the relevant references.]

Comments 6: Please, add the scale bar in Figure 3, 4C/4E, 5, and 6 including the figure legend. 

Respose 6: We have added scale bars to all the magnified pictures.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, I appreciated your effort in improving the manuscript following my suggestions. Unfortunately though, this is lacking of accuracy and several of my comments were not used to improve the manuscript. For example: 

-Comment 1, 2, and 3: were meant to be used to improve your manuscript. Based on your answers, I am expecting you add a corresponding paragraph in the manuscript, especially in the discussion for comment 2. As I mentioned, it is totally surprising that you find COL17 expression in the dermal papilla cells. 

-Comment 4: was done but the english language of that paragraph is very poor

-Comment 7: was done but in 3.5 it is not clear which model was used and where the changes were seen and 3.8 was not changed

-Comment 9: discussion was not revised accordingly

Comments on the Quality of English Language

Some part of the text requires the improvements of a native speaker (see above). Also the added text in the abstract is very poor.

Author Response

-Comment 1, 2, and 3: were meant to be used to improve your manuscript. Based on your answers, I am expecting you add a corresponding paragraph in the manuscript, especially in the discussion for comment 2. As I mentioned, it is totally surprising that you find COL17 expression in the dermal papilla cells. 

Response: Thank you for your suggestions and positive feedback. In response, we have included a discussion on the detection of type XVII collagen in HFDPC cells in the Discussion section.

-Comment 4: was done but the english language of that paragraph is very poor

Response: We have polished this part of the content to make its expression more academic.

-Comment 7: was done but in 3.5 it is not clear which model was used and where the changes were seen and 3.8 was not changed

Response: In the 3.5 part, we did not perform any modeling on the cells. Instead, we directly added rhCOL17A1 to the normal cells. The results showed that the expressions of C-myc and CyclinD1 increased. The title of 3.8 has been revised to make its expression clearer and more explicit.

-Comment 9: discussion was not revised accordingly

Response: In response to your comment that the Discussion section was predominantly summative rather than analytical, we have have made some improvements. We conducted a more in-depth discussion of the entire research content in the discussion section. We contextualized and discussed our findings in relation to previous research. The experimental results demonstrated a degree of consistency with other studies, further confirming the scientific validity and reliability of this work. Concurrently，The section heading has been updated from "Discussion" to "Discussion and Conclusions" to align with the revised content.

Comments on the Quality of English Language

Some part of the text requires the improvements of a native speaker (see above). Also the added text in the abstract is very poor

Response: The manuscript has been revised by a professional native speaker editor. As for the part you mentioned, it has also been re-polished.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed my questions.

Author Response

We sincerely thank the reviewers for their insightful comments and constructive suggestions, which have significantly improved the quality of our manuscript.

Reviewer 5 Report

Comments and Suggestions for Authors

Dear Authos

 

The response was relatively improved. 

However, there was still something to fill in your revised manuscript.

1. The authors should provide the regulatory number of your institute for using animal model. 

2. The author should provide 3 numbers for each gel image in the Western blot data

This was the most important thing, because the authors provided only 1 or 2 images. 

Othewise, the author violated the guideline of MDPI journal, and will be rejected. 

Author Response

Comments1:The authors should provide the regulatory number of your institute for using animal model. 

Response1: We have provided regulatory number for using animal model.

Comments2:The author should provide 3 numbers for each gel image in the Western blot data. This was the most important thing, because the authors provided only 1 or 2 images. Othewise, the author violated the guideline of MDPI journal, and will be rejected. 

Response2: Thank you for your dedication and rigor in supporting this study. We have reorganized the Western blot (WB) results and supplemented them with the calculated grayscale values. As you are well aware, while some WB bands might not appear significantly different upon visual inspection, significant differences in grayscale values become evident after normalization using β-actin. And the average values of the three images also showed a clear trend of effect. Based on the results of image grayscale value calculations, we consider the effect of rhCOL17A1 at the cellular level to be clear.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

My enquiries were now finally addressed

Reviewer 5 Report

Comments and Suggestions for Authors

Dear Authors

 

The authors responded to the reviewer's feedback adequately and your revised manuscript was relatively improved. ,