Knockdown of AKT isoforms and mTOR inhibition synergistically inhibits HCC cell proliferation. (A) shRNA mediated knockdown of single AKT isoform in HCC cell lines, confirmed by Western blot. (B) Hep3B knockdown cells were treated with 100 nM RAD001, 1.7 μM MK-2206, the combination of both, or DMSO as control, over 24 h, and mTOR and AKT signaling pathway activity was analyzed by Western blot. HSC70 served as loading control. AKT isoform knockdown cells were treated with 100nM RAD001 over 24 h. (C) Cells transduced with non-target vector were also treated with 1.7 μM MK-2206 or a combination of 100 nM RAD001 and 1.7 μM MK-2206. Proliferation was analyzed by BrdU incorporation. Columns, mean of one experiment performed in triplicates, bars, SD. *, p < 0.01; **, p < 0.001.