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. 2017 Mar 30;8(3):e2707. doi: 10.1038/cddis.2017.128

Figure 6.

Figure 6

miR-127 enhances myogenic differentiation by targeting S1PR3. (a) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of S1PR3 fold overexpression in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid; cells transfected with empty vector served as a negative control (NC). The data were normalized using GAPDH. S1PR3 expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. (b) Immunostaining for MyoG (green) in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid and NC cells transfected with empty vector. The cells were cultured in DM for 24 h. DAPI staining (magenta) indicates nuclei. Scale bars, 50 μm. (c) Quantification of MyoG+ cells in panel b. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed, and data are presented as positive cell numbers per view. (d) Quantification of MyoG expression by qRT-PCR in cells described in panel b. The data were normalized using GAPDH. MyoG expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. (e) Immunostaining for MHC (green) in miR-127 OE cells transiently transfected with an S1PR3 expression plasmid and NC cells transfected with empty vector (NC). The cells were cultured in DM for 48 h. DAPI staining (magenta) indicates nuclei. Scale bars, 50 μm. (f) The fusion index, calculated as the percentage of the total nuclei present in multinucleated myotubes, was determined for MHC-stained cells presented in panel e. The data are representative of three independent experiments. For each experiment, 10 representative views were analyzed. (g) Quantification of MHC expression in cells indicated in panel (e). The data were normalized using GAPDH. MHC expression levels were further normalized to the expression level of NC transfected with empty vector, defined as 1. NC, C2C12 cells infected with control lentivirus; OE, C2C12 cells infected with miR-127-OE lentivirus. Values represent means±S.E. of at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001