DU145 and PC3 cells are less sensitive to apoptosis than C42Luc cells.

Abstract

<p><b>A</b>) Treatment with combination of cycloheximide and ZSTK474 triggers MCL-1 loss in PC3, C42Luc, and DU145 cells, as well as BAD dephosphorylation in PTEN-deficient PC3 and C42Luc cells. Cell lysates prepared 6 hours after treatments were probed for pS112BAD, total BAD, and MCL-1, using β-actin as a loading control. Apoptosis in C42Luc, PC3, and DU145 cells was assessed by (<b>B</b>) measuring caspase 3 activity with fluorogenic substrate or (<b>C</b>) by time lapse microscopy. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. <b>D</b>) Knockdown of MCL-1 and ectopic expression of phosphorylation-deficient BAD were sufficient to induce apoptosis in prostate cancer cells. PC3 or DU145 cells were co-transfected with MCL-1-specific shRNA or scrambled control shRNA in combination with either a BAD2SA or WT-BAD expression construct. 48 hours post-transfection, apoptosis was analyzed by time-lapse microscopy. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields.</p