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

<p><b>A.</b> 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>B.</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. <b>C.</b> 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. <b>D.</b> 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). <b>E.</b> LH86 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g004" target="_blank">Figure 4D</a> and apoptosis was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g002" target="_blank">Figure 2A</a>. Statistical analysis was performed for apoptosis ratio by counting the number of cells with apoptotic nuclei (*p<0.05). <b>F.</b> LH86 cells treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g004" target="_blank">Figure 4D</a> 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.