Conformational changes of Bax during drug-induced apoptosis precede caspase activation.

Cells from a representative CLL patient were incubated with medium alone or the FCM combination in the presence or absence of 200 μM Z-VAD.fmk for 24 hours. Z-VAD.fmk was preincubated for 1 hour prior to the addition of FCM. In FCM-treated cells, a shift in the signal of fluorescence 2 (585 nm) and 3 (630 nm) was observed owing to the incorporation of mitoxantrone. The percentage of positive cells is indicated. Flow cytometric dot plots show cell viability as determined by annexin V binding (panel A); loss of ΔΨ_m and ROS generation by dual staining with DiOC₆[3] and DHE (panel B); quantification of the active form of caspase-3 (panel C); and conformational changes of Bax as determined by staining with anti-Bax (clone YHT-6A7) (panel D).