Evaluation of Teneligliptin and Retagliptin on the Clearance of Melanosome by Melanophagy in B16F1 Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, Kim et al. investigated melanophagy triggered by teneligliptin or retagliptin. However, the manuscript should be improved.

[1] The "Autophagy Analysis" and "Melanophagy Assay" subsections do not accurately introduce the measurement of autophagy and melanophagy. Autophagy is a dynamic process. When we talk about autophagy, we mean functional autophagy, and autophagic flux has to be measured. Measurement of GPF-LC3 puncta alone, LC3II alone, or LC3II/LC3I ratio cannot be used to measure autophagy and make a conclusion on autophagy-related roles. Autophagic flux has to be measured by using a lysosomal inhibitor (bafilomycin A1, chloroquine, etc.). Here, I use bafilomycin A1 as an example. If more cells with GFP-L3 puncta (or, more GFP-LC3 puncta per cell) were observed in the presence of bafilomycin A1 than in the absence of bafilomycin A1 and higher LC3II protein level was observed in the presence of bafilomycin A1 than in its absence, a positive autophagic flux and therefore functional autophagy exist in the type of cells studied. In this case, autophagy levels can be compared by comparing the number of cells with GFP-LC3 puncta (or the number of GFP-LC3 puncta per cell) or the LC3II protein levels in the presence of bafilomycin A1. Similarly, a lysosomal inhibitor must be used to examine the degradation of an autophagy substrate, such as p62. Figure 1C shows that functional autophagy exists in the tested cells, and teneligliptin hydrobromide (TGN) or retagliptin phosphate (RGN) can induce autophagy. However, the results of Figure 1C should be repeated by at least two more independent experiments.

[2] Figures 1A, 1B, 2, 3, 4, and 5 must be re-done by adding a lysosomal inhibitor (e.g., bafilomycin A1). Melanophagy should be analyzed by using a lysosomal inhibitor, an upstream inhibitor of autophagy (e.g., 3-methyladenine), and knockdown/knockout of at least two essential autophagy genes. If autophagy inhibition leads to increased levels of melanin, melanin is degraded by autophagy, and melanophagy exists.

 [3] Figure 2A shows that cell viability was changed by less t than 5% by drug treatment. The change is in the error range. Thus, the results are not meaningful.

 

[4] Protein band intensities in all western blots must be quantified, and the results must repeated by at least three independent experiments.

 [5] At least one piece of mechanistic information should be demonstrated in this study.

 [6] All experiments should be demonstrated in at least two cell lines.

 [7] English writing should be improved. Rules for scientific writing should be followed. For example, abbreviations should not be used in keywords. All abbreviations should be defined when first used in the abstract and the main text and constantly used thereafter. Past tense should be used to describe the experiments.

 [8] The manuscript writing should be improved. For example, the Introduction section should mention the gap in the literature, the objectives of the study, and the study's significance; the last two sentences in this section are conclusions and should not be included in this section.

Comments on the Quality of English Language

English writing should be improved. Rules for scientific writing should be followed. For example, abbreviations should not be used in keywords. All abbreviations should be defined when first used in the abstract and the main text and constantly used thereafter. Past tense should be used to describe the experiments.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a thorough study of chemical substances that alter the degradation of melanosomes by autophagy. Teneligliptin and retagliptin were identified in a screening and evaluated for their ability to modulate melanophagy. The design of the study is appropriate for the aims. The text is mostly well written. The figures are of good quality. I have of only minor comments.

 

As a note of caution, the authors should discuss the potential weaknesses of their experimental system. For example, they did not use primary human melanocytes but a mouse melanoma cell line. Although this choice is understandable for reasons of efficiency, the regulation of melanophagy may differ between a cell line and primary cells.

 

Can the authors comment in more detail on the stage of melanosome maturation that is primarily targeted by autophagy? In other words, does autophagy degrade melanosomes when they are still in the process of formation, or does it degrade mature melanosomes with high contents of melanin? If the latter is the case, by which biochemical mechanism is melanin degraded?

 

Autophagy is implicated in the control of skin aging, and aging is often associated with aberrant pigmentation. For the readership of the journal, more information on the role of autophagy in aging would be of great interest. The authors should mention reviews of autophagy in skin aging and discuss whether their findings are useful for cosmetic applications in aged skin.

 

Besides cosmetics, the text should also address the potential use of enhancers of melanophagy in pathological hyperpigmentation.

 

Line 62: add the granular layer

 

Several typos should be corrected, for example “maker protein” in line 53.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors Kim et al. in their ms titled “Evaluation of teneligliptin and retagliptin on the clearance of melanosome by melanophagy in B16F1 cells” in this studies showed that the essential insights into cellular degradation mechanisms and offers potential therapeutic avenues in the regulation of pigmentation. Over all the manuscript is well written and supported their conclusion based on their experimental results. However, I have few concern before acceptation.

Minor Comment:

1. Densitometry analysis for the WB is missing.

2. Three representative WB data add to the supplementary section.

3. The author only shown the changes in the protein expression level, what about the gene level.

Author Response

the author response is attached

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I appiciate the authors efforts for the revised version. Till I have few concern before acceptation. 

1. Check the Figure 1 & 2. It form layers of each other.

2. I did not find 3 separate representative WB images in the suuplimentary section. How author calculated the signifcance in densitometric analysis based on 1 WB images.

3. Although I agree to the author about their wrok related to ATG protein, but till they need to analysis the mRNA expression by qRTPCR. As both mRNA and protein expression are related to each other.

 

Author Response

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Author Response File: Author Response.pdf