Fig. 4.
Effect of wild-type GSK-3β overexpression and recombinant M-cadherin–Fc treatment on apoptosis induced by cell confluence or serum starvation. (A) A wild-type GSK-3β plasmid (GSK-3βWT) or an empty vector (EV) were transiently transfected into C2C12 myoblasts that were 80% confluent. 48 hours after transfection, cells were harvested in RIPA buffer and processed for immunoblotting for GSK-3β and cyclin D1 protein abundance. The experiment was repeated three times under each experimental condition. (B) Wild-type GSK-3β plasmids (GSK-3βWT) or empty vectors (EV) were transiently transfected into C2C12 myoblasts growing in recombinant M-cadherin–Fc-coated (M-cad-Fc) or vehicle-coated (Vehicle) dishes. 48 hours later, the cells were harvested in RIPA buffer and immunoblotted against pro-apoptotic proteins. GAPDH was probed as a loading control. (C) Densitometric analyses of immunoblot bands described in B. The data were normalized to GAPDH and expressed as the mean ± s.e.m. of three independent experiments. *P<0.05 vs EV/Vehicle or EV/M-cad-Fc. †P<0.05 vs GSK-3βWT/Vehicle. (D) Mitochondria were isolated from C2C12 myoblasts that had undergone the same treatments as described in B. The mitochondria were stained with JC-1. A FACSCalibur system was used to measure the change in mitochondrial membrane potential (Δψm). The data are expressed as the ratio of orange to green JC-1 staining. The data are reported as the mean ± s.e.m. of three independent experiments. *P<0.05 vs EV/Vehicle or EV/M-cad-Fc. †P<0.05 vs GSK-3βWT/Vehicle. (E) C2C12 myoblasts transfected with the wild-type-GSK-3β plasmid (GSK3βWT) or the empty vector (EV) and the non-transfected normal control (NC) cells were serum-starved for 0 hours (SS-0h), 6 hours (SS-6h), 12 hours (SS-12h), 24 hours (SS-24h) or 48 hours (SS-48h). At the end of each time point, the attached cells were harvested and DNA fragmentation was measured by a cell death ELISA assay. The data represent the mean ± s.e.m. from three independent experiments. *P<0.05, vs NC or EV. (F) The wild-type GSK-3β plasmid (GSK3βWT) or the empty vector (EV) was transiently transfected into C2C12 myoblasts growing in recombinant M-cadherin–Fc-coated (M-cad-Fc) or vehicle-coated (Vehicle) dishes. 48 hours later, the cells were treated with serum starvation for another 48 hours before being harvested. A cell death ELISA assay was used to measure the DNA fragmentation in the harvested cells. The data represent the mean ± s.e.m. from three independent experiments. *P<0.05 vs EV/Vehicle or EV/M-cad-Fc. †P<0.05 vs GSK-3βWT/Vehicle.