Effect of GSK-3β inhibition on cell-confluence-induced apoptosis.

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Effect of GSK-3β inhibition on cell-confluence-induced apoptosis. Effect of GSK-3β inhibition on cell-confluence-induced apoptosis. (A) C2C12 myoblasts were grown to 80% confluency then transfected with an empty vector (EV) or GSK-3βK85R, which contained a V5 tag (left panel), or constitutively active Akt (myrAkt) (right panel) for 48 hours. Non-transfected cells normal control cells (NC) were cultured under identical conditions. The protein abundances of V5 tag, GSK-3β, and cyclin D1 were examined in cells transfected with GSK-3βK85R plasmid (left panel) and those of Ser437-phosphorylated and total Akt were examined in myrAkt plasmid-transfected cells (right panel) by immunoblotting. (B) C2C12 myoblasts were grown to 80% confluency then co-transfected with the M-cadherin-targeted siRNA (M-) plus myrAkt (M-/myrAkt) or the GSK-3βK85R plasmid (M-/GSK-3βK85R) or an empty vector (M-/EV). Similar co-transfections were completed with the non-targeted scramble siRNA (SiCON) with myrAkt (SiCON/myrAkt), GSK-3βK85R (SiCON/GSK-3βK85R) or the empty vector (SiCON/EV). 48 hours after transfection, the cells were harvested and processed for immunoblotting of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. GAPDH was used as a loading control. Each experiment was repeated three times. (C) Densitometric analyses of immunoblots were obtained from C2C12 cells with identical treatments as described in Fig. 5B. The data represent the mean ± s.e.m. of three independent experiments. *P<0.05 vs SiCON/EV. †P<0.05 vs M-/EV. Yan Wang et al. J Cell Sci 2011;124:3835-3847 © 2011.