CRISPR-Mediated POSTN Editing Modulates Proliferation, Apoptosis, and Molecular Profiles of Primary Rabbit Hair Follicle Stem Cells via the cAMP/PKA/CREB Signaling Pathway

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents an interesting investigation of POSTN function in primary rabbit hair follicle stem cells using CRISPR/Cas9 editing, functional assays, transcriptomics, metabolomics, and integrated multi-omics analysis. The combined experimental approach is potentially valuable. However, several aspects of the experimental design, knockout validation, statistical analysis, and interpretation require clarification or additional validation before the conclusions can be considered sufficiently supported.

 

1. The title and several statements in the Abstract describe POSTN as a “key regulatory” factor in hair growth. However, the study evaluates proliferation and apoptosis only in cultured rabbit HFSCs and does not directly examine hair-shaft elongation, hair-cycle progression, follicle regeneration, or in vivo hair growth. In addition, the Abstract states that POSTN maintains proliferation–apoptosis balance through coordinated regulation of gene expression and metabolism, although the multi-omics results establish associations rather than direct mechanisms. The authors are kindly encouraged to limit these claims to HFSC proliferation, survival, and molecular homeostasis or provide a direct hair-growth model. A more precise title could be “CRISPR-Mediated POSTN Depletion Alters Proliferation, Apoptosis, and Molecular Profiles of Primary Rabbit Hair Follicle Stem Cells.”
2. In section 2.1, the total number of rabbits and the number of independent HFSC isolations are not reported. This information is essential because it is unclear whether n=3 represents cells isolated from three different rabbits or technical replicates derived from one animal. Please report the number of animals, allocation of samples, housing conditions, hair-cycle stage, and whether each biological replicate originated from an independent donor. The authors should also describe postoperative analgesia, wound closure, animal monitoring, and the fate of the animals after full-thickness skin collection. The anesthetic name “Zoteil-50” should also be verified and corrected if necessary.
3. In section 2.2, the cell-isolation procedure is described only briefly, and important details such as tissue digestion, follicle microdissection, filtration, cell-selection strategy, seeding density, and culture duration are missing. Please provide a sufficiently detailed protocol to allow reproducibility. In addition, the experimental control appears to consist of untreated wild-type cells. Because plasmid transfection, Cas9 expression, GFP sorting, and post-sorting recovery may independently influence proliferation, apoptosis, and stress-related pathways, a non-targeting sgRNA control should undergo the same transfection, GFP sorting, and culture procedures as the POSTN-targeted group. This matched control is important for distinguishing POSTN-specific effects from transfection- or CRISPR-associated cellular stress.
4. The reported editing efficiency is based on Sanger sequencing of only 15 TA-cloned PCR products for each sgRNA. This approach estimates the frequency of edited amplicons but does not demonstrate biallelic functional knockout in individual HFSCs. In-frame mutations, monoallelic editing, and residual wild-type cells may remain in the population. The authors are encouraged to quantify the allele distribution using amplicon deep sequencing or another validated quantitative method and report the proportions of frameshift and in-frame mutations. Residual POSTN should also be evaluated at the intracellular, secreted, and extracellular-matrix-associated levels. Until complete functional loss is demonstrated, the terms “POSTN-edited” or “POSTN-depleted” would be more accurate than “POSTN knockout.”
5. In proliferation and apoptosis assay, CCK-8 measures cellular metabolic activity rather than proliferation directly, which is especially relevant because POSTN editing produced substantial metabolic alterations. Therefore, reduced CCK-8 absorbance may reflect reduced metabolic activity, reduced cell number, or both. The authors are encouraged to include direct viable-cell counting, cell-cycle analysis, or a colony-formation assay. Similarly, TUNEL detects DNA fragmentation but is not entirely specific for apoptosis. Confirmation using Annexin V/propidium iodide flow cytometry and cleaved caspase-3 or cleaved PARP analysis would strengthen the apoptosis conclusion. Please also report the numbers of cells and microscopic fields analyzed and whether image acquisition and quantification were performed blindly.
6. The manuscript states that comparisons were performed using independent two-tailed Student’s t-tests. However, the CCK-8 experiment includes repeated measurements at 0, 24, 48, and 72 hours and should be analyzed using a two-way repeated-measures analysis or a mixed-effects model that evaluates treatment, time, and treatment-by-time interaction. Multiple genes and proteins are also evaluated without a clearly described correction for multiple comparisons. Please report exact P-values, effect sizes, confidence intervals, biological replicate numbers, and technical replicate numbers. Individual biological data points should be displayed whenever possible.
7. The Discussion appropriately acknowledges that pathway enrichment indicates statistical association rather than direct activation. Nevertheless, later statements conclude that POSTN regulates MAPK, PI3K–Akt, cAMP, AMPK, FoxO, glucagon, and glycerophospholipid pathways. These conclusions should be moderated unless supported experimentally. The authors may select one biologically plausible pathway, such as integrin/FAK/PI3K–Akt or integrin/FAK/ERK, and validate total and phosphorylated pathway proteins. Without such validation, the omics findings should be presented as candidate mechanisms for future investigation.

 

Minor suggestion

8. Several references should be re-evaluated to ensure that they directly support the corresponding statements. For example, Reference 26 concerns FGF signaling in the chicken primitive streak and does not directly support hair-follicle transition from telogen to anagen. Reference 24 examines human dermal papilla cells rather than HFSCs, and Reference 35 is a review of dermatological effects of GLP-1 receptor agonists rather than direct evidence for glucagon signaling in the hair-follicle microenvironment. Please replace these references with direct, cell-type-specific, and mechanistically relevant evidence where available.
9. Section 4, HFSCs are described as “pluripotent stem cells.” Adult HFSCs are generally considered multipotent rather than pluripotent. Please revise this terminology throughout the manuscript.
10. The terms “knockout,” “knockdown,” “POSTN deficiency,” and “POSTN group” are used inconsistently. Please select terminology that accurately reflects the experimental model and apply it consistently. If complete biallelic loss is not demonstrated, “POSTN-edited” or “POSTN-depleted” would be preferable.
11. Please correct the typographical error “ReSponse” to “Response.” Gene and protein nomenclature should also be standardized, with gene symbols distinguished clearly from protein names.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors isolated primary hair follicle stem cells from male Angora rabbits for POSTN knockout. They performed transcriptomic profiling and metabolomic analysis to reveal the effects of POSTN's absence.

Major concerns:

1. After editing, the authors did not isolate single clones to obtain strains with an identical genetic background. Meanwhile, all subsequent experiments were performed on a mixed population with different indels, which raises significant concerns about variation within the cell population.
2. Because of the non-identical genetic background, the cell population is closer to a "knockdown" than a "knockout." The description of the bulk sequencing data raises further concerns about signal dilution due to cellular heterogeneity and poor reproducibility. In Fig. 4A, the PCA data showed clustered WT samples but dispersed POSTN samples, which already demonstrates the bias among the POSTN-edited groups.
3. The authors cannot even rule out that the observed changes stem from off-target effects in this scenario.
4. The manuscript as a whole is plain and superficial. It merely sequences the so-called "knockout" mixture and provides a brief, very basic and general profiling. I believe that establishing a "key regulatory role of POSTN in hair growth" requires a much more comprehensive approach than simply listing genes and signaling pathways. The sequencing data should serve as a tool to help identify the most relevant candidates for further experimental validation, either ex vivo or in vivo.

Minor concerns:

1. Both SOX9 and LHX2 are transcription factors. Fig. 1A fails to clearly show that either SOX9 or LHX2 is localized exclusively to cell nuclei. The magnification and image resolution need to be improved.
2. Fig. 3 is titled "POSTN Deficiency Suppresses the Biological Functions of HFSCs," and Section 3.3 is titled "POSTN Deficiency Inhibits the Biological Functions of HFSCs." However, both the figure and the corresponding paragraph discuss "development-, proliferation-, and apoptosis-related genes." There is no assay testing cellular functions specific to hair follicle stem cells' own nature, such as their stemness. These subtitles are too broad and imprecise.

It is understandable that primary tissue cells such as hair follicle stem cells may not be easy to single-clone. If that is the case, a CRISPRi or even a knockdown system may work better in this scenario, since neither introduces double-strand breaks the way a CRISPR/Cas9 knockout vector does.

The manuscript is not recommended for publication in its current form.

Comments on the Quality of English Language

The language part is acceptable, however, the scientific part should be improved.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

comments

• create a more attractive title
• provide a figure demosntraing the periostin - hair shaft interactions
• are there any studies on periostin and hair loss disorders? report in intro
• provide references of each section of the methods you reported
• provide larger images in the text or better organixe them
• you found altered metabolic and immunomodulatey pathways - make plausible connection with hair disorders scuh as alopecia areata
• add a limitations section 
• general nice study with well analysed methods section 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has revised the manuscript as per suggestion, which has further strenghten its quality. 

Author Response

Thank you very much for the reviewers ' recognition and affirmation of the revised manuscript. The suggestions you put forward are of key value to improve the quality of the manuscript. Thank you again for your careful guidance and professional suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed most of my previous comments. However, there are still several important points requiring clarification that need to be resolved before publication.

The authors should be careful if using AI to generate the illustration of the molecular mechanism in Figure 9:

1. The CRISPR/Cas9 protein complex embraces the open DNA helix (two strands) and produces a double-strand break at the same time. The illustration is misleading and inaccurate because (1) Cas9 only holds one strand, and (2) a redundant pair of scissors labeled as sgRNA - Cas9 (blue sphere) functions as a second pair of scissors.
2. The FACS sorting part is a mess. It is neither precise nor straightforward.
3. There are at least five different colors of bubbles presented in Metabolomics, but only two colors are labeled with text for categories. It is unclear whether the depth of color represents any parameter.
4. What are T197 and S133 next to p-PKA and p-CREB? They did not appear anywhere within the main body text.
5. The illustration has an embedded title with the symbol “”, which is not a correct format.
6. Please standardize the fonts and color codes within Figure 9.

It leaves too many AI traits without proper polish. Don’t ruin this manuscript at the very end. Please pay attention to the publication requirements if an AI tool was used, and provide a proper citation.

Author Response

Thank you very much to the reviewers for their careful review and valuable professional corrections. In view of the series of problems such as inaccurate molecular mechanism indication, unclear experimental process presentation, and non-standard format labeling in the mechanism diagram of figure 9, we have completely abandoned the original version, and re-based on the experimental process of this study and the verified molecular mechanism conclusions. The whole picture 9 was independently drawn, which strictly conforms to the presentation standards of academic publishing and fully meets the publishing requirements of journals. Please review the correction and thank you again for your rigorous and professional guidance, which effectively helps us to improve the standardization and scientificity of manuscripts.

Reviewer 3 Report

Comments and Suggestions for Authors

the authors considered my suggestions- fig 9 if is AI cretaed better remove it

Author Response

Thanks to the reviewers for their recognition and valuable suggestions. In view of the relevant reminders of Figure 9, we have completed the rectification according to your suggestions. Based on the experimental conclusions and core logic of this study, we have re-drawn the schematic diagram of the mechanism independently, and completely replaced the original Figure 9 to ensure that the charts are all original and in line with academic norms. Thank you again for your rigorous check, please review.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

I have no further questions.