Fig 2: Up-regulation of MRTF-A and PAX7 during muscle cell differentiation and muscle regeneration. (A) MRTF-A and PAX7 presented the same expression patterns during satellite cells (SCs) differentiation. The qPCR assay was conducted using sorted SCs, SCs maintained in growth medium (GM), and SCs at days 1, 2 or 3 of differentiation in differentiation medium (DM). (B) The mRNA expression of the MRTF-A and PAX7 genes in differentiating C2C12 cells. (C) Haematoxylin-eosin stain was performed to assess the muscle repair after CTX injection for 1, 5, 7 and 10 days. NS represented the normal saline-treated group. Scale bar, 50 µm. (D) The MRTF-A mRNA expression in NS control and muscle tissue after CTX injection for 0, 1, 3, 5, 7, 10 and 14 days. (E) The PAX7 mRNA expression in NS control and muscle tissue after CTX injection for 0, 1, 3, 5, 7, 10 and 14 days. (F) The protein level of the MRTF-A and PAX7 in CTX-injected tissues. (G) The densitometric quantification analysis of three independent Western blot experiments. The ß-actin was used to serve as a loading control. **p < 0.01

Fig 3: RNA-seq results reveal MRTF-A knockdown down-regulates/up-regulates the skeletal muscle regulation involving genes in C2C12 cells. (A) The mRNA and protein levels of MRTF-A in C2C12 cells transfected with shRNA lentivirus targeting MRTF-A as compared to the shRNA-control group. (B) Volcano plot of differentially expressed genes between sh-MRTF-A and shRNA-control in C2C12 cells as determined by RNA-seq. (C) Statistics of GO Enrichment analysis to categorize the pathways that are significantly altered upon MRTF-A knockdown. The striated muscle thin filament, skeletal muscle contraction, regulation of muscle contraction and muscle contraction signalling are highlighted. (D) The heat map analysis showing the differentially regulated skeletal muscle development genes, data were presented as log2(FPKM+1). (E) Validation of identified muscle cell self-renewal–related genes through FPKM (Reads Per Kilobase of exon model per Million mapped reads, FPKM =1) in MRTF-A knockdown or control muscle cells. (F) Validation of identified muscle cell differentiation related genes through FPKM. *p < 0.05, **p < 0.01

Fig 4: MRTF-A regulates PAX7 expression by directly binding to the CArG box region of the PAX7 promoter. (A) Promoter activity of the PAX7 gene with WT, Cut-1 (CArG box 1 cut-down), Cut-2 (CArG box 2 cut-down), Mutation-1 (CArG box 1 mutation) and Mutation-2 (CArG box 2 mutation) promoters by dual-luciferase reporter assay. (B) The effects of MRTF-A on promoter activity of the PAX7 gene. The pCDH-vector, pCDH-MRTF-A, shRNA-control and shRNA-MRTF-A were co-transfected with dual-luciferase reporter plasmids, respectively. (C) The effects of MRTF-A on promoter activity of the PAX7 gene with Cut-1 or Cut-2 promoter. The pCDH-vector, pCDH-MRTF-A, shRNA-control and shRNA-MRTF-A were co-transfected with Cut-1 or Cut-2 plasmid, respectively. (D) Detection of interaction of MRTF-A and CArG box 1 or CArG box 2 within the PAX7 promoter by EMSA. A complete set of three reactions was performed using the nuclear extracts prepared from normal C2C12 cells. The 200-fold of unlabeled probes were used as specific competitors to demonstrate that the signal shift observed results from specific protein: DNA interaction. Arrowhead shows the specific complex. (E) The binding of the MRTF-A on CArG box of the PAX7 gene promoter by ChIP assay. Histone H3 Antibody treatment was used as the positive control. (F) The percentage of input in IgG and MRTF-A antibody–treated groups. *p < 0.05, **p < 0.01

Fig 5: MRTF-A inhibits the differentiation of myoblast. (A) The mRNA expression of MRTF-A, PAX7, MyoD and MyoG in C2C12 cells transfected with pCDH-vector or pCDH-MRTF-A. (B) Western blot for MRTF-A, PAX7, MyoD and MyoG in C2C12 cells transfected with pCDH-vector or pCDH-MRTF-A. (C, D) The mRNA and protein level of MRTF-A, PAX7, MyoD and MyoG in C2C12 cells transfected with shRNA-control and shRNA-MRTF-A. The densitometric quantification was analysed from three independent Western blot experiments (B, D). GAPDH expression was analysed to ensure equal loading of samples. (E) The differentiation of C2C12 cells stably overexpressing MRTF-A was examined by staining for MyoG after 0 d (D0) and 3 d (D3) of culture in DM. Cells with an empty vector as the control. (F) The graph shows MyoG-positive cells as a proportion of total cell number (shown by DAPI staining) for MRTF-A-overexpressing cells compared to control cells. (G) MyHC immunocytochemistry (red) for MRTF-A overexpressing cells and control cells (pCDH-vector) at D0 and D3 of differentiation. (H) Number of fibres per field of view and number of nuclei per fibre in images used for counting in (G), shown relative to control cells, *p < 0.05, **p < 0.01