Figure 4.

Silencing of c-Met in PC-3 and DU145 cells though c-Met small interfering RNA (siRNA) transfection. (A and B) PC-3 and DU145 cells were transfected with c-Met and scramble siRNA (100 nM) for 24 h using electroporation. The transfected cells were pre-treated with EVO (5 µM) for 4 h and then treated with HGF (50 ng/mL) for 15 min. Then, equal amounts of whole cell lysate were analyzed by Western blot using antibodies against phospho-c-Met(Tyr1234/1235) and c-Met. After overnight incubation in serum-free conditions following transfection, the cells were pre-treated with EVO (5 µM) for 4 h and then treated with HGF (50 ng/mL) for 15 min in serum-free conditions. Then, equal amounts of whole cell lysate were analyzed by Western blot using antibodies against p-Src and p-STAT3. (C and D) Transfected PC-3 and DU145 cells were re-seeded in 96-well plates (5 × 10³ cells/well) and pre-treated with EVO (5 µM) for 1 h, then treated with HGF (50 ng/mL) or left untreated for 48 h. Cell viability was compared after the different treatments using the MTT assay. (E and F) Transfected cells were pre-treated with EVO (5 µM) for 1 h then treated with HGF (50 ng/mL) or left untreated for 36 h. The cells were fixed, stained with TUNEL assay reagent, and then analyzed by flow cytometry. NT and EVO (−)/HGF (−) indicate control samples treated with medium containing 0.1% DMSO.