FIG. 7.

Characterization of puma- and caspase-independent cell death. WT (A) and puma^−/− (B) neurons were transfected with Smac-YFP and 24 h posttransfection loaded with TMRM (20 nM) in experimental buffer and mounted on the thermostatically regulated stage of a Zeiss 5Live confocal microscope. Neurons were treated with epoxomicin (50 nM), and images were acquired every 5 min. (C) Quantification of the percentage of WT and puma^−/− neurons where Δψ_m loss occurred in the absence of Smac release. Smac release was taken as reduction in the standard deviation (S.D.) of Smac-YFP fluorescence (n = 26 and n = 15 cells for WT and puma^−/− neurons, respectively, from at least five independent experiments; *, P < 0.05 for comparisons as assessed by Fisher's exact t test). (D) Duration of Δψ_m loss was measured in WT neurons undergoing FRET disruption and puma-deficient neurons in which disruption was absent (n = 15 for WT and n = 16 for puma^−/− cells from at least 10 independent experiments; *, P < 0.05). (E) Images illustrating type I and type II apoptosis. puma^−/− neurons were transfected with Smac-YFP and 24 h posttransfection loaded with TMRM (20 nM) and Hoechst (1 μg/ml) in experimental buffer. Neurons were treated with epoxomicin (50 nM), and images were acquired in real time every 5 min. Scale bar, 10 μm. (F) Images illustrating the lack of nuclear fragmentation in puma^−/− neurons. WT and puma^−/− neurons were treated with epoxomicin (50 nM) and stained with Hoechst 24 h posttreatment. Scale bar, 15 μm.