Figure 4

Effects of MEK1/2 inhibitor U0126 or PD98059, p38 kinase inhibitor SB203580, JNK inhibitor SP600125 and PI-3 kinase inhibitor LY294002 on cell viability, MAPK, Akt-1, phosphorylation of MAPK (Thr202/Tyr204), phosphorylation of Akt-1 (Ser473), p38 kinase, phosphorylation of p38 kinase (Tyr182), JNK, phosphorylation of JNK (Thr183/Tyr185), cytochrome c release, and cleavage of caspase 3, caspase 7 and PARP in HepG2 cells. Cells were grown and treated with 0.1% of DMSO (C) or 10 μM of U0126 (U0) or 10 μM of LY294002 (LY) or 50 μM of PD98059 (PD) or 5 μM of SB203580 (SB) or 10 μM of SP600125 (SP) in phenol red free serum free MEM (PSF) medium for 48 h as described under Experimental Procedures. Total cell lysate (for detection of all proteins except cytochrome c) or mitochondria free cytosol (detection of cytochrome c) from HepG2 (A) cells was subjected to Western blot analysis as described under Experimental Procedures. Blots containing total cell lysate were incubated with mouse anti-α-tubulin, mouse anti-phospho-MAPK (Thr202/Tyr204), rabbit anti-Akt-1, rabbit anti-phospho Akt-1 (Ser473), rabbit anti-p38, rabbit anti-phospho-p38 (Tyr182), rabbit anti-JNK1/2, rabbit anti-phospho-JNK1/2 (Thr183/Tyr185), rabbit anti-caspase-3, rabbit anti-cleaved caspase-7 (20 kDa), rabbit anti-cleaved PARP antibodies. Blots containing mitochondria free cytosol were blotted with mouse anti-cytochrome c antibody. All the antibodies were used at a final concentration of 1 μg per ml. (B) Cell viability of HepG2 following different treatments was analyzed by MTT assay as described under Experimental Procedures. Bars with different letters are significantly different from one another at (p < 0.01) as determined by ANOVA test. The results represent the mean of 3 experiments ± SE is shown.