Figure 2.

Induction of apoptosis following combination treatment of bufalin with MK2206 was associated with the inhibition of the AKT/mTOR pathway. (a) H929 cells were transfected with an AKT shRNA (H929 Sh-AKT) and/or a non-targeting shRNA (H929-NC) and protein lysates were subjected to Western blot analysis using antibodies specific against p-AKT and β-actin. (b) H929-NC and H929 Sh-AKT cells were incubated with 0, 3, 6, 12 nM of bufalin for 48 h. Cell viability was measured by CCK8 assay, whereas the cell cycle distribution was determined by flow cytometry analysis of the DNA content and cell apoptosis by the AnnexinV/PI assay. Each bar represented the mean±S.E. of triplicate experiments. (c) H929 and U266 cells were treated with 12 nM of bufalin in the absence and/or presence of 6 μM of MK2206 for 24 and 48 h, and the levels of the phosphorylated and total AKT, mTOR, P70 and 4EBP1 proteins were examined by western blot analysis. β-actin was used as a loading control. The values under the bands were representative of the mean quantitation ratios compared with the control groups. Experiments were performed in triplicate (*P<0.05; **P<0.01)