Fig. 3. Inhibition of GSK-3 blocked ceramide-induced mitochondrial apoptosis. 10I cells were treated with 25 µM of C₂-ceramide for 6 hours with or without the GSK-3 inhibitors LiCl (10 and 20 mM) and SB216763 (10 and 20 µM), or pre-transfected for 24 hours with control siRNA (100 nM) or GSK-3 siRNA (50 and 100 nM). (A) We used Rhodamine 123 staining plus flow cytometry to detect the _m reduction. Partial histograms are shown. The percentages of cells with _m reduction are given as the average of triplicate cultures (mean ± s.d.). (B) We used western blotting to determine the cytosolic levels of cytochrome c. -actin levels were determined as an internal control. (C) We used enzymatic cleavage of the specific substrates benzyloxycarbonyl-Leu-Glu(-OMe)-His-Asp(-OMe)-pNA and benzyloxycarbonyl-Asp(-OMe)-Glu(-OMe)-Val-Asp(-OMe)-pNA to determine the activities of caspase-9 and caspase-3. Data are given as the average of triplicate cultures (mean ± s.d.). pNA, p-nitroanilide.