Myostatin Regulates Inflammatory Cytokine and Chemokine Expression, Rheumatoid Arthritis Synovial Fibroblast Invasion, and CD4+ Th Cell Transmigration
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe Authors address the topic of myostatin supporting pro-inflammatory processes in rheumatoid arthritis. The topic is definitely of interest but the research article requires an improvement for easier reading and understanding.
For a better data presentation I suggest a model such as https://doi.org/10.1038/s41467-025-57002-6
The following issues should be addressed:
Line 17- FN-beta?
Lines 19-20- “20 ng/mL, 24 h” are not pro-inflammatory mediators
The unnecessary repetitions should be avoided (e.g. under section 2. Materials and Methods- the name of manufactures are mentioned once the reagents are first presented, the manufacturer’s name should not be all over the text where the reagents are mentioned).
The use of one single denomination will help the flow of the text- RPMI-1640 with 10% FBS and 1% penicillin-streptomycin was used for the culture of two different cell lines (MH7A and CD4+Th):
Lines 83-85- RPMI-1640 with 10% FBS and 1% penicillin-streptomycin
Line 92- the same RPMI-1640 with 10% FBS and 1% penicillin-streptomycin becomes T cell medium
Line 119- the manufacturer’s name should not be there
Line 155- it should be “in a 5% CO2 incubator”
Lines 262-263 The phrase requires revision.
Fig 3C FN-gamma?
Section 2 Materials and Methods 2.3 ELISA- It should be mentioned if the final values are the averages of measurement performed in duplicates or triplicates.
Section 3 Results rows 218-219- It should be explained how the proliferation rate was calculated and how the count of cells per hour was done.
Line 236 “n=3” It should be mentioned “n=3 separate experiments”
Line 249 “24 h post-treatment”- Was the treatment removed and the cells kept in culture 24 h more?
Lines 251- 258- A comparison between 24 and 48 h treatments will give a better view of the results. It should be explain how the fold difference was calculated.
Lines 274-276 The statement should be statistically supported
Lines 276-278 To state this 24 and 48 h treatments need to be compared.
Lines 286-287 An insert with the fold difference might be useful.
Fig 1A, 1B, Fig 6A, 6B and 6C It should be explain what means time point 0 hrs. Were the cells treated for 0 hrs?
Fig 3 line 303 n=3 Please explain what 3 means.
Fig 5 line 337 n=3 Please explain what 3 means.
Fig 6 line 365 n=3 Please explain what 3 means.
Section 3.6 lines 375- 381- explanations for Figs 7 B, C, E, F are confused. Please revised the text.
Lines 378-380 –Compared to what is the fold decrease of CD4+ Th cell transmigration?
Author Response
We appreciate your thoughtful and detailed review, and we tried our best to strengthen the manuscript based on your comments.
Comment 1: Line 17- FN-beta?
Response 1: FN-beta is corrected to ‘IFN-beta’ (Line 17).
Comment 2: Lines 19-20- “20 ng/mL, 24 h” are not pro-inflammatory mediators
Response 2: ‘20 ng/mL and 24 h’ are removed (Lines 19-20)
Comment 3: The unnecessary repetitions should be avoided (e.g. under section 2. Materials and Methods- the name of manufactures are mentioned once the reagents are first presented, the manufacturer’s name should not be all over the text where the reagents are mentioned).
Response 3: When the same reagent is mentioned, the manufacturer's name is not mentioned except for the first-mentioned reagent. When the manufacturer’s name repeats, the city and country names of the manufacturer are removed to prevent unnecessary repetitions.
Comment 4: The use of one single denomination will help the flow of the text- RPMI-1640 with 10% FBS and 1% penicillin-streptomycin was used for the culture of two different cell lines (MH7A and CD4+Th):
Lines 83-85- RPMI-1640 with 10% FBS and 1% penicillin-streptomycin
Line 92- the same RPMI-1640 with 10% FBS and 1% penicillin-streptomycin becomes T cell medium
Response 4: To achieve a single denomination, we corrected the section as follows: MH7A cells, immortalized human RASF, were obtained from the Riken Cell Bank, Ibaraki, Japan (RCB1512: MH7A). Primary CD4+ Helper T Cells (CD4+ Th) were obtained from the American Type Culture Collection (Cat. no: PCS-800-016, ATCC, VA, USA). MH7A and CD4+ Th cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640, ATCC modification, Thermo Fisher Scientific, PA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin/ streptomycin (Gibco, MT, USA) (Lines 83-89).
Comment 5: Line 119- the manufacturer’s name should not be there
Response 5: The manufacturer’s name (R&D Systems) is removed (Lines 121-122).
Comment 6: Line 155- it should be “in a 5% CO2 incubator”
Response 6: We corrected it. (Line 158).
Comment 7: Lines 262-263 The phrase requires revision.
Response 7: The phrase is revised as follows: Treated cell pellets were used to synthesize cDNA and subsequent RT-qPCR experiments. Data in the figures are represented as mean ± SEM (n = 3 independent measurements) (Lines 327- 329).
Comment 8: Fig 3C FN-gamma?
Response 8: FN-γ is corrected into IFN-γ ( Figure 3C).
Comment 9: Section 2 Materials and Methods 2.3 ELISA- It should be mentioned if the final values are the averages of measurements performed in duplicates or triplicates.
Response 9: We added the averages of the measurements, which read as follows: All ELISA experiments were performed according to the manufacturer’s instructions, with triplicate measurements for all samples (Lines 129-131).
Comment 10: Section 3 Results rows 218-219- It should be explained how the proliferation rate was calculated and how the count of cells per hour was done.
Response 10: To calculate the proliferation rate per hour, 24 h cell numbers were divided by 24. We realized that this calculation was inappropriate to examine the per-hour proliferation rate. Thus, we removed Figure 1C and revised the statement. The following is the revised statement. ‘When we examined the number of RASF and HSF cells at 48 h, RASF exhibited significantly higher cell counts compared with HSFs' (Lines 231-232).
Comment 11:Line 236 “n=3” It should be mentioned “n=3 separate experiments”
Response 11: We corrected it as it was pointed out (Line 268).
Comment 12: Line 249 “24 h post-treatment”- Was the treatment removed and the cells kept in culture 24 h more?
Response 12: The 24 h post-treatment was an incorrect statement. We changed the 24 h post-treatment to 24 h treatment (Line 281).
Comment 13: Lines 251- 258- A comparison between 24 and 48 h treatments will give a better view of the results. It should be explain how the fold difference was calculated.
Response 13: mRNA expression was determined using the 2-ΔΔCt method. For each sample, the Cq value of the target gene was normalized to that of GAPDH housekeeping to generate a ΔCt value (ΔCt = Cq(Target) – Cq(GAPDH)). Next, the mean ΔCt of untreated control samples was determined. The ΔΔCt was then obtained by subtracting the average control ΔCt from the ΔCt of each individual sample (ΔΔCt = ΔCt(Sample) – ΔCt(Control)). Finally, the fold change in mRNA expression relative to the control was expressed as 2–ΔΔCt
Since the relative mRNA expression at 0 h is close to 1, the fold difference can be estimated from the histograms in Figure 2.
Comment 14: Lines 274-276 The statement should be statistically supported
Response 14: The statement was not based on statistical analysis but on the visual difference in the histograms. Thus, we removed the statement in Lines 274-276 in the original manuscript.
Comment 15: Lines 276-278 To state this 24 and 48 h treatments need to be compared.
Response 15: We think that the histograms of Figure 3 show the statistical significance of 24 and 48 h treatments.
Comment 16: Lines 286-287 An insert with the fold difference might be useful.
Response 16: We found that statements in lines 283-287 in the original manuscript were not clearly expressed, thus corrected as follows: To determine whether MSTN has similar effects in HSFs, we measured the secretion of TNF-a, IL-17, IFN-g, and CXCL1 after 24 h MSTN treatment (0, 10, and 20 ng/mL) using ELISA. Relative to RASFs, HSFs exhibited markedly reduced cytokine and chemokine secretion. At 20 ng/mL MSTN, TNF-α, IL-17, IFN-γ, and CXCL1 secretion in HSFs was approximately 10-, 37-, 11-, and 35-fold lower, respectively, than that observed in RASFs. (Figures 4A–D),(Lines 345-349)
Comment 17: Fig 1A, 1B, Fig 6A, 6B and 6C It should be explain what means time point 0 hrs. Were the cells treated for 0 hrs?
Response 17:0 h means the starting point of the RASFs and HSFs culture with different concentrations of MSTN. We revised the legends for Figures 1A and 1 B as follows: (A) RASF cells and (B) HSF cells were treated with MSTN (0, 10, and 20 ng/mL) at 0 h, and cells were counted at 0, 24, and 48 h. We also revised the legends of Fig 6a, 6 B, and 6C as follows: (A) RASF cells were treated with MSTN (0, 10, or 20 ng/mL) at 0 h and cell pellets were collected at 0, 24, and 48 h, then used for RT-qPCR to measure MMP-3 mRNA expression. (B) Culture supernatants were used to measure MMP-3 secretion by ELISA. (C) HSFs were similarly treated with MSTN (0, 10, or 20 mg/mL) at 0 h, and supernatants were collected at 0, 24, and 48 h to measure MMP-3 secretion by ELISA.
Comment 18: Fig 3 line 303 n=3 Please explain what 3 means.
Fig 5 line 337 n=3 Please explain what 3 means.
Fig 6 line 365 n=3 Please explain what 3 means.
Response 18: We corrected them
Comment 19: Section 3.6 lines 375- 381- explanations for Figs 7 B, C, E, F are confused. Please revised the text.
Lines 378-380 –Compared to what is the fold decrease of CD4+ Th cell transmigration?
Response 19: We revised the text as follows: We also examined the involvement of IL-17 and CXCL1 in CD4 Th cell transmigration by blocking the activities of IL-17 and CXCL1 using neutralizing antibodies against the molecules. MSTN stimulation significantly increased CD4 Th cell transmigration dose-dependently in the Control, IL-17 blocked, and CXCL1 blocked CCM (Figures 7A-F). Blocking IL-17 or CXCL1 significantly decreased the CD4 Th cell transmigration compared to control (Figures 7A-C and Supplementary Table 14), suggesting that IL-17 and CXCL1 may play a role in regulating trans migration. The decrease of CD4 Th cell transmigration by blocking CXCL1 was much greater than that of blocking IL-17 (Figure 7G), underscoring the predominant role of CXCL1 in mediating CD4Th cell transmigration (Lines 514-523).
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this paper the authors examined the role of myostatin (MSTN) in the induction of proinflammatory cytokines in rheumatoid arthritis synovial fibrocytes (RASF). Also, they studied whether MSTN promotes RASF proliferation, RASF invasion and CD4 T cell migration. All experiments were done in vitro using a transformed cell line, MH7A cells, and for control healthy fibroblast-like synoviocytes ((HFS).
The authors convincingly show that MSTN induces expression and production/secretion of all 11 cytokines and chemokines that have been examined, in RASF. Production of TNF-α, IL-17, IFN-γ, and CXCL1 after MSTN treatment of HFS was several times lower than production of these cytokines by MSTN stimulated RASF. MSTN promotes RASFs invasion and CD4 T cell migration
MSTN did not promote RASF or HFS proliferation, but induced CD90 (Thy-1) expression. Of interest is their finding that IFN-γ and IL-17 upregulated MSTN expression. Based on these findings the authors postulate that MSTN and some inflammatory cytokines may establish a vicious cycle that upregulates joint inflammation.
Weaknesses:
- The authors claim that their findings “offer new insights into the role of MSTN in RA pathogenesis”. This is an overstatement, in fact all the work is done in vitro and the RASF are a transformed cell line. Therefore, no such postulate should be presented. In contrast the authors should comment on the use of a transformed cell line that may be useful for indicative results.
- Expression of CD90 should be done by FACS analysis, the way that is done, (presented in figure 1) do not allow to draw quantitative conclusions.
- The paper needs some polishing. For example: line 63, 64 “MSTN enhanced the production 63 of TNF-α expression”
Line 68 “severity in model mice for human RA”
Linne 130 “Cell pellets were lysed with NP-40 lysis buffer (150 mM NaCl, 1% Triton X-100, 50 130 mM Tris, pH 8.0)
P values should be corrected and use the same symbols in all figures.
Author Response
We thank you for your time, thoughtful review, and valuable input. We tried our best to strengthen the manuscript based on your comments.
In this paper the authors examined the role of myostatin (MSTN) in the induction of proinflammatory cytokines in rheumatoid arthritis synovial fibrocytes (RASF). Also, they studied whether MSTN promotes RASF proliferation, RASF invasion and CD4 T cell migration. All experiments were done in vitro using a transformed cell line, MH7A cells, and for control healthy fibroblast-like synoviocytes ((HFS).
The authors convincingly show that MSTN induces expression and production/secretion of all 11 cytokines and chemokines that have been examined, in RASF. Production of TNF-α, IL-17, IFN-γ, and CXCL1 after MSTN treatment of HFS was several times lower than production of these cytokines by MSTN stimulated RASF. MSTN promotes RASFs invasion and CD4 T cell migration
MSTN did not promote RASF or HFS proliferation, but induced CD90 (Thy-1) expression. Of interest is their finding that IFN-γ and IL-17 upregulated MSTN expression. Based on these findings the authors postulate that MSTN and some inflammatory cytokines may establish a vicious cycle that upregulates joint inflammation.
Weaknesses:
Comment 1: The authors claim that their findings “offer new insights into the role of MSTN in RA pathogenesis”. This is an overstatement; in fact all the work is done in vitro and the RASF are a transformed cell line. Therefore, no such postulate should be presented. In contrast the authors should comment on the use of a transformed cell line that may be useful for indicative results.
Response 1: We corrected this (Lines 644- 646).
Comment 2: Expression of CD90 should be done by FACS analysis, the way that is done, (presented in figure 1) do not allow to draw quantitative conclusions.
Response 2: We did not perform FACS analysis; instead, CD90⁺ cells were identified by immunofluorescence staining and quantified by cell counting. Figures 1D and 1F present the numbers of CD90⁺ RASFs and HSFs, respectively.
Comment 3: The paper needs some polishing. For example: line 63, 64 “MSTN enhanced the production 63 of TNF-α expression”
Response 3: We have revised this statement: MSTN promoted TNF-α production through activation of the PI3K–Akt pathway (31) and increased IL-1β expression by suppressing miR-21-5p in RASFs. (32) (Lines 63-65).
Comment 4: Line 68 “severity in model mice for human RA”
Response 4: We have revised this statement: MSTN deficiency or antibody-mediated blockade significantly ameliorated arthritis severity in murine models of human RA (Lines 67-68).
Comment 5: Linne 130 “Cell pellets were lysed with NP-40 lysis buffer (150 mM NaCl, 1% Triton X-100, 50 130 mM Tris, pH 8.0)
Response 5: The following is the statement in line 133-134 in the revised manuscript: Cell pellets were lysed with NP-40 lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris, pH 8.0)
Comment 6: P values should be corrected and use the same symbols in all figures.
Response 6: We corrected the p-values to be consistent.
Reviewer 3 Report
Comments and Suggestions for AuthorsIntroduction: elaborate the specific objectives (experiments) in the last paragraph, and mention the comparison of the RASFs with the HSFs
Methods: 2.1 Cell Cultures- mention the size of the tissue culture dishes and the starting cell counts of each of the cell types (RASFs & HSFs) for the experiments. 2.8 Add Data Analysis to the title, and list the analysis of the data for all of the relative representation of data.
Results:
Figure 1c. delete it, or show the starting cell count of both cell types
Figure 2. change the Y-axis to target mRNA expression and place the unit/relative in brackets (fold of respective control). Add a note in the legend that the respective controls are the respective time points
Figure 3. place the name of the cytokine/chemokine (target) on the Y-axis and place the unit (pg/ml) in bracket below the name of the target
Figure 4. A-D: illustrate the results of the HSFs separately, and show the results of the data corresponding to Figures 2 &3 for the HSFs; or clarify the rationale for doing so. Figure 4 E-H. change the Y-axis title and unit to clarify the relative quantitation
Figures 5 & 6. place or explain the lack of mRNA expression data for HSFs, and change the Y-axis relative to list what the data is relative to.
Figure 7G. edit the Y-axis title/unit
Figure 8b. change the title of the Y-axis to cell invasion and place the OD in bracket, to indicate the unit. Figure 8c. change the Y-axis title invaded RASFs and in bracket (% of migrated cells)
Discussion: discuss separately the rationale/results using the RASFs & the HSFs; and then the comparison of the effects
Author Response
We thank you for your time, thoughtful review, and valuable input. We tried our best to strengthen the manuscript based on your comments.
Comment 1: Introduction: elaborate the specific objectives (experiments) in the last paragraph, and mention the comparison of the RASFs with the HSFs
Response 1: We added the following statement in the last paragraph of the Introduction section in the revised manuscript: The objectives of this study were to investigate the role of MSTN in regulating the expression of key inflammatory cytokines and chemokines, to examine the reciprocal effects of these mediators on MSTN expression in RASFs, and to determine the impact of MSTN on RASF invasion and CD4⁺ Th cell transmigration, with comparative analysis between RASFs with the HSFs (Lines 76-80).
Comment 2: Methods: 2.1 Cell Cultures- mention the size of the tissue culture dishes and the starting cell counts of each of the cell types (RASFs & HSFs) for the experiments. 2.8 Add Data Analysis to the title, and list the analysis of the data for all of the relative representation of data.
Response 2: We added the following statements
2.1 Cell Cultures
RASFs and HSFs cells were cultured in sterile, flat-bottomed, 24-well polystyrene plates (MilliporeSigma, MA, USA). Each well provided a growth area of approximately 1.9 cm2. Cells were seeded at a density of 300,000 cells per well, with 300 µL of complete growth medium added to each well (Lines 92-95).
2.8 Data analysis
mRNA expression levels were quantified using the 2-ΔΔCt method, normalized to the GAPDH housekeeping gene. ELISA data were analyzed by generating standard curves using a non-linear curve fitting, four-parameter logistic regression, according to the manufacturer’s instructions (Quantikine ELISA, R&D Systems, MN, USA). Western blot band intensities were quantified and normalized to GAPDH. For quantification of cell transmigration and invasion results, stained membranes were imaged and analyzed in ImageJ using a fixed pipeline (8-bit conversion, rolling-ball background subtraction, binary cleanup, watershed) and Analyzed Particles. Threshold parameters and a macro were applied uniformly to all images. All statistical analyses were performed using R (R Core Team, version 4.1.3, Vienna, Austria) and GraphPad Prism version 10.2.3 (MA, USA). Data in the figures represent mean ± SEM (n = 3). Group comparisons were performed using the Nonparametric Kruskal-Wallis rank sum test, followed by Dunn’s multiple comparison test with p-values adjusted by the Holm method. Group differences significant at 0.1, 0.05, and 0.01 were denoted by †(p < 0.1), *(p < 0.05), and **(p < 0.01), respectively (Lines 209-224).
Results:
Comment 3: Figure 1c. delete it, or show the starting cell count of both cell types
Response 3: We realized that the calculation of the proliferation rate per hour was inappropriate to examine the per-hour proliferation rate. Thus, we removed Figure 1C and revised the statement. The following is the revised statement. ‘When we examined the number of RASF and HSF cells at 48 h, RASF exhibited significantly higher cell counts compared with HSFs' (Lines 231-232).
Comment 4: Figure 2. change the Y-axis to target mRNA expression and place the unit/relative in brackets (fold of respective control). Add a note in the legend that the respective controls are the respective time points
Response 4: We revised Figure 2 with a proper Y-axis title with units and added a note in the figure legend.
Comment 5: Figure 3. place the name of the cytokine/chemokine (target) on the Y-axis and place the unit (pg/ml) in bracket below the name of the target
Response 5: We revised Figure 3 with a corrected Y-axis title
Comment 6: Figure 4. A-D: illustrate the results of the HSFs separately, and show the results of the data corresponding to Figures 2 &3 for the HSFs; or clarify the rationale for doing so. Figure 4 E-H. change the Y-axis title and unit to clarify the relative quantitation
Response 6: We did not perform ELISA for all 11 cytokines/chemokines in HSFs due to time constraints, availability of resources, and cost; instead, we selected four for analysis. Accordingly, the figures present a comparison between RASFs and HSFs for these four cytokines. Figure 4E–H has been revised with corrected Y-axis titles.
Comment 7: Figures 5 & 6. place or explain the lack of mRNA expression data for HSFs, and change the Y-axis relative to list what the data is relative to.
Response 7: We did not perform mRNA expression experiments for HSFs due to time constraints, availability of resources, and cost. We did only ELISA experiments for HSFs. We believe these data sufficiently support our conclusions regarding HSF responses. We revised the Figures 5 and 6 with a proper Y-axis titles.
Comment 8: Figure 7G. edit the Y-axis title/unit
Response 8: We edited Figure 7G as suggested
Comment 9: Figure 8b. change the title of the Y-axis to cell invasion and place the OD in bracket, to indicate the unit. Figure 8c. change the Y-axis title invaded RASFs and in bracket (% of migrated cells)
Response 9: We revised Figures 8b and c with corrected Y-axis titles
Comment 10: Discussion: discuss separately the rationale/results using the RASFs & the HSFs; and then the comparison of the effects
Response 10: We have made a concerted effort to clearly separate the rationale and results for RASFs and HSFs in the Discussion section, while also comparing their responses to highlight the distinct effects observed in each cell type. We believe this revision improves the clarity and flow of the manuscript.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors fulfilled almost all the points raised by the reviewer.
Regarding the answer to Comment 17, there is a misunderstanding and misconception about the meaning of “time 0”. By any meanings “time 0” is NO-treatment, it is the time right before adding the treatment reagents. The authors are showing in Figs 1A, 1B, 6A, 6B and 6C a “time 0 treatment”. The only real “time 0” is 0 ng/ml MSTN. Once the reagents were added (10 or 20 ng/ml MSTN) there is a treatment time (seconds, minutes, hours). If the authors want to keep the display of the graphs for Figs 1A, 1B, 6A, 6B and 6C as it is they should state for how long time (seconds, minutes etc) was the treatment for what they call “time 0”.
The article does not have an abbreviation section. It might be useful to have one.
Also, the authors should be aware that in their reply the lines are not properly mentioned (e.g. Response 16 (lines 345-349) actually the lines are 332-337).
Author Response
We appreciate the reviewer’s insightful comments and suggestions.
Comment 1: Regarding the answer to Comment 17, there is a misunderstanding and misconception about the meaning of “time 0”. By any meanings “time 0” is NO-treatment, it is the time right before adding the treatment reagents. The authors are showing in Figs 1A, 1B, 6A, 6B and 6C a “time 0 treatment”. The only real “time 0” is 0 ng/ml MSTN. Once the reagents were added (10 or 20 ng/ml MSTN) there is a treatment time (seconds, minutes, hours). If the authors want to keep the display of the graphs for Figs 1A, 1B, 6A, 6B and 6C as it is they should state for how long time (seconds, minutes etc) was the treatment for what they call “time 0”.
Response 1: RASFs or HSFs were primarily treated with MSTN 0, 10, and 20 ng/mL (R&D Systems, MN, USA) at room temperature for 30 minutes. The cell counts were measured after 0, 24, and 48 h incubation. In Figures 1A and 1B, “time 0h” means directly after treating RASFs and HSFs with MSTN at room temperature for 30 minutes.
After treating cells with MSTN for 30 minutes, the cell pellets and the supernatants were separately collected after 0, 24, and 48 h incubation by centrifuging at x10,000 g for 10 minutes for ELISA and RT-qPCR experiments. In figures 6A,6B, and 6C, “time 0h” means directly after treating cells with MSTN at room temperature for 30 minutes.
We corrected this in section 2.1 Cell cultures as, “RASFs or HSFs were primarily treated with MSTN 0, 10, and 20 ng/mL (R&D Systems, MN, USA) for 30 minutes at room temperature. The cell cultures were incubated for 0, 24, and 48 h. Following the incubation, cell counts were measured by the TC20 automated cell counter (Bio-Rad, CA, USA). The cell pellets and supernatants were collected separately after centrifugation at 10,000 x g for 10 minutes. The cell pellets were used in Real-time PCR and Western blot assays, and the cell culture supernatants were used in ELISA assays” (Lines 96- 102 in the second revised manuscript). We also revised the legends of Figures 1A,1B, and 6A,6B, and 6C as follows. “Figure 1. MSTN did not affect the proliferation of RASF or HSF cells. (A) RASF cells and (B) HSF cells were primarily treated with MSTN (0, 10, and 20 ng/mL) for 30 minutes, and cells were counted at 0, 24, and 48 h.(The 0 h counts of RASFs and HSFs treated with MSTN were performed within 30 minutes)".
“Figure 6. No cross-stimulation between MSTN and MMP-3 in RASFs and HSFs. (A) RASF cells were primarily treated with MSTN (0, 10, or 20 ng/mL) for 30 minutes and cell pellets were collected at 0, 24, and 48 h, then used for RT-qPCR to measure MMP-3 mRNA expression. (B) Culture supernatants were used to measure MMP-3 secretion by ELISA. (C) HSFs were similarly treated with MSTN (0, 10, or 20 mg/mL) for 30 minutes, and supernatants were collected at 0, 24, and 48 h to measure MMP-3 secretion by ELISA. (The 0 h experiments of RASFs and HSFs treated with MSTN were performed within 30 minutes)”.
Comment 2: The article does not have an abbreviation section. It might be useful to have one.
Response 2: We added an abbreviation section.
RA Rheumatoid Arthritis
SFs Synovial fibroblasts
RASFs Rheumatoid arthritis synovial fibroblasts
HSFs Healthy synovial fibroblasts
MSTN Myostatin
IL Interleukin
TNF Tumor necrosis factor
IFN Interferon
CXCL C-X-C motif chemokine ligand
CCL C-C motif chemokine ligand
MMP Matrix metalloproteinase
TGF-β Transforming growth factor beta
CD Cluster differentiation
THY-1 Thymocyte antigen-1
Th T helper cells
CCM Conditioned culture medium
Comment 3: Also, the authors should be aware that in their reply the lines are not properly mentioned (e.g. Response 16 (lines 345-349) actually the lines are 332-337).
Response 3: We apologize for the confusion regarding the line references in our previous response. We have carefully reviewed and mentioned the correct line numbers in the second revised manuscript to ensure they correspond accurately. We appreciate the reviewer’s attention to detail, which has helped us improve the clarity of our revisions.
