Figure 5. Endothelin-1 induces activation of PI3K-AKT-GSK pathway in mouse myoblasts (C2C12) through ETA receptor and ROS production. (A, B) Cells were incubated with 1 nM ET-1 at different times. Activation of PI3K-AKT-GSK pathway was analyzed by Western blot measuring phosphorylation of AKT (P-AKT, panel A) and phosphorylation of GSK (P-GSK, panel B). (C, D) Cells were incubated in the presence of different antagonists to block PI3K-AKT-GSK pathway (Wortmannin: 10 μM WTN; AKT inhibitor: 30 μM I-AKT), to block ET receptors (ETA receptor antagonist: 100 nM BQ123; ETB receptor antagonist: 100 nM BQ788) (C), and to block ROS production (antioxidant N-acetylcysteine: 100 μM NAC) (D). All of them were added at least 30 min before 1 nM ET-1 for 24h, to assay P-AKT (closed bars) or P-GSK (open bars). Representative Western blots are shown at the top of each panel. The densitometric analysis is shown below of each panel. Values are the mean±SEM of 5 independent experiments, *p<0.05 vs. control cells (C), and **p<0.05 vs ET alone.