BimEL knockdown by shRNA prevents conformational change of Bax/Bak and apoptosis in cells coexposed to PD184352/UCN-01. (A) U226 cells were stably transfected with constructs encoding shRNA against human Bim (shBim) or scrambled shRNA (pSUPER) as control, respectively, after which stable clones (1 for pSUPER control and 2 for shBim, designed W4 and W12) were obtained by selection with puromycin. Western blot analysis was performed to monitor down-regulation of Bim expression in both shBim clones (inset). Cells were then exposed to 100 nM UCN-01 plus or minus 5 μM PD184352 for 40 hours, after which the percentage of dead cells (7-AAD+) was determined by 7-AAD staining and flow cytometry. (B) Parallel studies were performed in RPMI8226. 8226/pSUPER and 8226/shBim cells were treated with 150 nM UCN-01 plus or minus 5 μM PD184352 for 24 hours, and the content of dead cells was determined. For panels A and B, the results represent the means (± SD) for 3 separate experiments performed in triplicate. **Significantly lower than the value for pSUPER control cells with same treatment (P < .001 for both panels). (C) U266/shBim and U266/pSUPER cells were treated as described for panel A, after which cells were lysed and subjected to Western blot analysis to assess phospho-ERK1/2 expression, caspase-3 cleavage, and PARP degradation. CF indicates cleavage fragment. Each lane was loaded with 20 μg protein; blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer. Two additional studies yielded equivalent results. (D) Alternatively, cells were lysed and subjected to immunoprecipitation (IP) with Bax 6A7 or Bak Ab-1 and then WB using anti-Bax or anti-Bak to monitor the conformational change of Bax and Bak as described for Figure 3C; 200 μg protein per condition was employed for IP. IgG levels are shown to ensure equal loading and transfer. The results of representative experiments are shown; 2 additional studies yielded equivalent results.
