Figure 4. Survivin down-regulation sensitizes ABT-263-induced apoptosis in HCC cells.

A. LH86 cells were treated as indicated and cell lysates were prepared for Western blotting. Pro-apoptotic proteins: Bax, Bad, and Bak and anti-apoptotic proteins Bcl-xL and Mcl-1 were assessed with specific antibodies respectively. β-actin was detected and served as an equal protein loading control. B. LH86 cells were untreated or treated with ABT-263 (1 µM), YM-155 (1 µM) or combination of ABT-263 (1 µM) and YM155 (1 µM) for up to 6 h as indicated. Then cells were harvested and cell lysates were prepared for Western blotting. Anti-survivin and anti-Bcl-xL polyclonal antibodies were used to assess protein levels for survivin and Bcl-xL respectively. β-actin was used as an equal protein loading control. The band intensities of survivin, Bcl-xL, and β-actin was qualified with Image J software. C. LH86 cells were transiently transfected with synthesized random siRNA (control) or survivin specific siRNA duplexes, and 48 h post-transfection, cells were subjected to Western blotting analysis with anti-survivin polyclonal antibody. β-actin was used as an equal protein loading control. D. LH86 cells were transfected with synthesized random control siRNA or survivin specific siRNA, and 48 h post-transfection, cells were untreated or treated with ABT-263 (1 µM) for 24 h and then subjected to Hoechst staining to show apoptotic cells with condensed nuclei (representative apoptotic cells were marked with white arrows). E. LH86 cells were treated as in Figure 4D and apoptosis was measured as in Figure 2A. Statistical analysis was performed for apoptosis ratio by counting the number of cells with apoptotic nuclei (*p<0.05). F. LH86 cells treated as in Figure 4D were harvested and cell lysates were prepared and subjected to Western blotting. Apoptosis was determined through caspase 3 activation. β-actin was used as an equal protein loading control.