Figure 6. Role of PI3K-AKT-GSK pathway in endothelin-dependent cellular fibrosis and cellular senescence. Cells were incubated in the presence of different antagonists to block PI3K-AKT-GSK pathway (Wortmannin: 10 μM WTN; LY-294,002 hydrochloride: 50 μM LY; AKT inhibitor: 30 μM I-AKT). All of them were added at least 30 min before adding 1 nM ET-1 for 24h to assay FN expression by Western blot (panel A) or by Immunofluorescence (panel B), and to assay senescence by measuring p16 protein content for 48h by Western blot (panel C) and SA-ß-GAL activity for 72h (panel D). Representative Western blots are shown at the top of panels (A, C). Representative microphotographs are shown on the top of panel B, D with 40x magnification, scale bar, 50 μm. The densitometric analysis is shown below of each panel. Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells (C), and **p<0.05 vs ET alone.