Fig. 3. Apoptotic alterations induced by Nbk in Bax-expressing DU145 clones. DU145 clones expressing exogenous Bax were generated by retroviral transfer of the Bax cDNA under control of the CMV promoter. The DU145-mock clone was generated by transduction with control HyTK retrovirus. Stable clones were transduced with Ad5-myc-Nbk-tTA and cultured for 24 h in the presence (Tet-off condition) or absence of doxycyclin (Tet-on condition). Control cells were mock treated and grown in the absence of doxycyclin. (A) Western blot analysis of Bax and Nbk expression, cytochrome c release and procaspase-9 processing in DU145-Bax clones 24 h after infection with Ad5-myc-NBK-tTA. (B) Disruption of mitochondrial membrane potential (Δψm) by mycNbk in DU145-Bax cells. Cells were incubated with JC-1, a cationic dye that exhibits potential-dependent accumulation in mitochondria, and fluorescence intensity was measured by flow cytometry. Upper panel: representative experiment. The percentage of cells with Δψm loss is indicated between markers. Lower panel: means ± SD of the percentages of cells with Δψm loss from three independent experiments.