FIGURE 4:

Inhibition of myogenesis by PLD1 silencing in L6 muscle cells. (A) L6 myoblasts were transfected with either pSuper-PLD1 shRNA (PLD1-shRNA) or luciferase shRNA (Luc-shRNA). Western blot analysis showed that PLD1 expression was significantly reduced by RNA interference in clones 1 and 2 compared with Luc-shRNA–treated cells and L6 myoblasts. (B) PLD1 knockdown inhibited PLD activity stimulated by PMA, whereas the basal activity was not affected (n = 3, *p < 0.05). (C, D) PLD1 knockdown resulted in delayed onset of myoblast fusion. Luc- and PLD1-shRNA myoblasts were cultured until nearly confluent and switched to low-serum differentiation medium. Myoblast fusion was monitored daily for 4 d using phase-contrast microscopy, and fusion index was analyzed at days 2 and 4. Data are shown as mean ± SD (n = 3, *p < 0.05).