Figure 3.

SMUL facilitates myoblast proliferation but suppresses myoblast differentiation in vitro

(A) Relative SMUL expression after introducing SMUL into CPMs (n = 4). (B) Relative mRNA levels of several cell cycle genes after overexpression of SMUL (n = 4). (C) Proliferation of transfected CPMs was assessed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation (n = 3). (D) Proliferation rate of myoblasts with SMUL overexpression (n = 3). (E) CCK-8 assays were performed in CPMs with SMUL overexpression (n = 6). (F) Cell cycle analysis of myoblasts at 48 h after overexpression of SMUL (n = 4). (G–I) MyHC immunostaining (G) (n = 3), myotube area (%) (H) (n = 3), and myoblast fusion index (I) (n = 3) of CPMs transduced with SMUL overexpression. Cells were differentiated for 72 h after transfection. The nuclei were visualized with DAPI. (J and K) Relative mRNA (J) (n = 4) and protein (K) (n = 3) expression levels of myoblast differentiation marker genes from pcDNA3.1-SMUL-transfected CPMs. The numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold change relative to the control. Results are shown as mean ± SEM. Statistical significances of differences between means were assessed using an independent sample t test. ∗p < 0.05, ∗∗p < 0.01.