Figure 2.

Silencing OIP5-AS1 attenuates myogenesis and inhibiting miR-7 promotes myogenesis. (A) AB678 myoblasts were transfected with Ctrl siRNA or OIP5-AS1-directed siRNA; 24 h later, they were placed in differentiation medium for 72 h, whereupon differentiation was monitored by assessing MYH levels (green) by immunofluorescence (left), and the fusion index and number of nuclei per myotube were quantified (right) after assessing five separate fields per experiment. (B) AB678 myoblasts were transfected with Ctrl siRNA or OIP5-AS1-directed siRNA; they were then placed in differentiation medium, and collected at the times shown after the induction of differentiation. The levels of MYOG, MYH and loading control HSP90 were assessed by western blot analysis. (C) AB678 myoblasts were transfected with Ctrl miR, miR-7 mimic or miR-7 antagomiR; they were then placed in differentiation medium for 72 h, and differentiation was monitored by assessing MYH levels (red) by immunofluorescence (left), and the fusion index and number of nuclei per myotube were quantified (right) after assessing five separate fields per experiment. (D) AB678 myoblasts were transfected with Ctrl miR, miR-7 mimic or miR-7 antagomiR; as described in panel (B), the levels of MYOG, MYH, MEF2C and loading control HSP90 were assessed by western blot analysis. (E) Cells were processed as described in panel (C), and the levels of CK activity were measured enzymatically (see the ‘Materials and Methods’ section). Data in all panels represent the means ± SEM from three or more biological replicates. Significance was established using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. In panels (B) and (D), bands were quantified by densitometry and the relative intensities are indicated.