Figure 5.

Immunofluorescence and protein analysis of the pro-apoptotic effect of sorafenib and BEZ235 on HepG2 and HepG2^R cells. A. Annexin V-FITC/propidium iodide staining and histograms showing the percentage of apoptotic cells in HepG2 and HepG2^R treated with sorafenib (HepG2, 4 μM; HepG2^R, 10 μM), BEZ235 (0.25 μM), and in combination for 24 h. Sorafenib and BEZ235 individually induced apoptosis of HepG2 and HepG2^R cells compared with the control, and the combination of two drugs had a greater effect than did the agents alone (**P<0.01). B. Total protein was extracted from HepG2 and HepG2^R cells treated with sorafenib (HepG2, 4 μM; HepG2^R, 10 μM), BEZ235 (0.25 μM), and in combination for 24 h. The amounts of apoptosis-related proteins were determined with western blot. The combination of sorafenib and BEZ235 promoted pro-apoptotic proteins caspase-9, caspase-3, and caspase-7 activation and up-regulated pro-apoptotic protein Bad, but not Bax or Bak. (***P<0.001; **P<0.01; and *P<0.05). Data shown are the mean ± SD from three independent experiments.