Figure 4.

Analysis of the effect of combined sorafenib and BEZ235 treatment on the cell cycle, as determined with flow cytometry and western blot. A. HepG2 and HepG2^R cells stained with propidium iodide after treatment with sorafenib (HepG2, 4 μM; HepG2^R, 10 μM), BEZ235 (0.25 μM), or combined agents for 24 h. Cell-cycle distribution was analyzed by flow cytometry. Compared with control values, sorafenib and BEZ235 blocked the cell cycle at the G0/G1 phase, and blocking was greater with the two drugs combined (P<0.05). B. Total protein was extracted from HepG2 and HepG2^R cells after treatment with sorafenib (HepG2, 4 μM; HepG2^R, 10 μM), BEZ235 (0.25 μM), or combined agents for 24 h. Western blot analysis revealed that the key G1-phase regulatory proteins, cyclin D1 and p-Rb, and their upstream molecule, p-GSK3β, were also down-regulated. (**P<0.01; and *P<0.05), which arrested cells at G0/G1 phase. Data shown are the mean ± SD from three independent experiments.