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. 2013 Sep 5;8(9):e74561. doi: 10.1371/journal.pone.0074561

Figure 3. Bim knockdown decreased sensitivity of C42Luc cells to apoptosis.

Figure 3

A) NOXA expression is decreased in C42Luc cells treated with the combination of ZSTK474 and cycloheximide. Western blot analysis of MCL-1, NOXA, BIM and β-actin (loading control) in cells treated for 4 and 6 hours with cycloheximide. B) Co-immunoprecipitation of MCL-1 and BIM from cells that expressed doxycycline-inducible FLAG-MCL-1. C) Apoptosis in C42Luc cells that express two BIM-specific shRNA and in parental cells was assessed by time lapse microscopy. Cells were treated with vehicle ((–) DMSO) or combination of cycloheximide and ZSTK474 (+). Amount of apoptosis (%) was determined as in Figure 1A. At least 100 cells were counted for each treatment. Error bars show standard deviations from the average of four randomly chosen fields. Data for 6 hours post-treatment are presented. Differences in% apoptosis between control cells (that express scrambled shRNA) and C42Luc BIM1 or C42Luc BIM2 cells were statistically significant (p<0.05 and p<0.0001, respectively). Insert shows decreased BIM expression in cells that stably expressed BIM-specific shRNA. D) Apoptosis in C42Luc cells that expressed one of two BIM-specific shRNA or control vector was assessed by measuring caspase activity. Cells were treated for 6 hours with vehicle ((–) DMSO) or the combination of cycloheximide and ZSTK474 (+). Differences in caspase activity between control cells and C42Luc BIM1 or C42Luc BIM2 cells were statistically significant (p = 0.002 and p = 0.05, respectively). E) C42Luc cells or C42LucBIM2 cells that stably express BIM-targeting shRNA were transfected with lentiviral vector that express GFP and either scrambled (scr) or MCL-1 –specific shRNA2. Percent of apoptosis in GFP-positive cells was determined by time-lapse microscopy. At least 100 cells were counted for each treatment. Error bars show average and standard error of four visual fields. Qualitatively similar results were obtained in C42LucBIM1 cells. Beta actin was used as a loading control in (A) and (C).