Fig. 4. Bax is required for Nbk-induced apoptosis. Stable Bax-expressing clones were transduced with Ad5-myc-Nbk-tTA as described in Figure 3 and cultured 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) Flow cytometric measurement of hypodiploid DNA. Upper panel: representative experiment. The percentage of apoptotic cells in a representative experiment was measured 24 h after adenoviral tranduction and is indicated between markers. Lower panel: means ± SD from three independent experiments. (B) Detection of phosphatidlyserine exposure: flow cytometric detection of apoptotic cells was performed by staining with annexin V–FITC to detect exposure of phosphatidylserine onto the cell surface 18 h after transduction. Cells were counterstained with propidium iodide (PI) to detect membrane damage. PI-positive cells were considered as necrotic and excluded from the analysis. The percentage of apoptotic cells is indicated between markers. Upper panel: representative experiment. Lower panel: means ± SD from three independent experiments.