Figure 5.

The combination of bufalin with Mk2206 surpassed bortezomib resistance in bortezomib-resistant cells. (a) H929R and U266R cells were treated with 24 nM of bufalin for 3, 6 and 12 h and subsequently subjected to Western blot analysis using anti-p-AKT, anti-AKT and anti-β-actin antibodies. 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. (b) H929R and U266R cells were treated with 24 nM of bufalin and/or 12 μM of MK2206 for 48 h, and the apoptotic rates were analyzed by Annexin V/PI assay. The combination group exhibited statistically different values compared with the treatment of bufalin and/or MK2206 alone. Each bar represented the mean±SE (standard error) of 3 independent experiments. (c) H929R and U266R cells were treated with 24 nM of bufalin in the absence and/or presence of 12 μM of MK2206 for 12, 24, 36 and 48 h and protein lysates were subjected to immunoblot analysis using antibodies specific against PARP, caspase-3, caspase-9 and β-actin. β-actin was used as a loading control. (d) H929R and U266R cells were treated with 24 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 (*P<0.05; **P<0.01)