Fig. 2.

Setdb1 depletion leads to downregulation of myogenic regulatory factors in proliferating C2C12 cells. (A) Exogenous Setdb1 not targeted by Setdb1 shRNA rescues myogenic differentiation in Setdb1-depleted myoblast cells. C2C12 cells depleted of endogenous Setdb1 were infected with retrovirus expressing Flag-Setdb1 which is not targeted by Setdb1 shRNA used for cell line establishment. Empty vector was used as a control. Differentiation was assessed by the appearance of myotubes after 72 h in serum-deprived growth medium (left) and expressions of MHC and MyoD (right). (B, C) Total RNAs were prepared from both control and Setdb1 shRNA-expressing C2C12 cells prior to or 72 h after initiation of differentiation. RNA was analyzed by real-time PCR using primers specific for MyoD (B) and myogenin (C). Results were quantified using the standard curve method and normalized to GAPDH. Data were represented as relative expression. Shown are representative data of at least three independent experiments performed in triplicate, and error bars indicate standard deviation. *p-value < 0.05 and **p-value < 0.01