Figure 4.

Combination of survivin knockdown with ABT-263 and trametinib effectively induces apoptosis of KRAS-mutant lung adenocarcinoma cells. (A) Effects of triple combination therapy of survivin knockdown, ABT-263 and trametinib on the viability of H358 and H441 cells. Cells were transfected with BIRC5 siRNA #1 or #2 (10 nM each) and cultured for 48 h. Cells were then treated with the ABT-263 (1 μM) alone, trametinib (25 nM) alone, both, or neither for a further 72 h. Results are shown as the means ± SD; ^**P<0.01. (B) Crystal violet staining of viable cells. NC siRNA or BIRC5 siRNA #1 (10 nM each)-transfected cells were grown for 48 h. A total of 5×10⁵ cells were then seeded in 6-well plates and treated with ABT-263 (1 μM) alone, trametinib (25 nM) alone, both, or neither for a further 72 h. Cells were then fixed and stained with crystal violet. (C) The effects of triple combination therapy of survivin knockdown, ABT-263 and trametinib on caspase 3/7 activity in H358 and H441 cells. The NC siRNA- or BIRC5 siRNA #1 (10 nM each)-transfected cells were cultured for 48 h, and the cells were then treated with ABT-263 (1 μM) alone, trametinib (25 nM) alone, both, or neither for a further 24 h before evaluating caspase activity. Caspase 3/7 activity was normalized to 100 for the mean of three control (NCsi) dishes. Columns, mean (n=3); bars, SD; ^**P<0.01. (D) Western blot analysis of the effects of the triple combination therapy on H358 and H441 cells. Cells were treated in the same manner as described in (A) or (B). Of note, Bim (EL) was dephosphorylated by trametinib treatment, and then accumulated in the cell. EL, extra-long; L, long; S, short.