Figure 6

Induction of Bax and Bak-independent cyt. c release, caspase activation and cell death by PAC-1. (a) The expression status of Bax and Bak protein in MEF WT and DKO cells analyzed by western blot. (b) Analysis of nuclear condensation after PAC-1(50 μM) treatment in MEF WT and DKO cells by Hoechst staining. Representative images and graph are shown (n=4, mean ±S.D.). MEF WT versus DKO, 12 h (P<0.0001) and 24 h (P<0.001). (c) Clonogenic survival was assessed in both cells upon PAC-1 treatment as described. Representative graph and images are shown (n=3, mean ±S.D.). MEF WT versus DKO (P<0.001). (d) Analysis of annexin V – PE staining in MEF WT and DKO cells by flow cytometry after treatment with PAC-1. Shifting of population towards right suggests the annexin exposure to cell surface. (e) MEF WT and DKO cells are transiently transfected with CFP-DEVD-YFP FRET probe and treated with PAC-1 for indicated time points. DEVD cleavage was detected by western blot after PAC-1 treatment by probing with GFP antibody. (f) Caspase activation was analyzed in MEFs cell by western blot upon PAC-1 treatment for different time points. Caspase-3, -7, -9, -2, -8 and Bid-specific antibodies were used. HSC-70 or β- actin served as loading control. (g) Cyt. c release was analyzed in cytosolic fraction of digitonin permeabilized MEFs cells by western blot after PAC-1(50 μM) treatment in time-dependent manner. (h) MEF DKO cells were transiently transfected with cyt. c-EGFP plasmid and imaged under fluorescent microscope after PAC-1 treatment for 24 h (scale bar: 20 μm). The diffuse pattern of cyt. c-EGFP in treated cells indicates its release from mitochondria to cytosol while granular pattern in untreated cells indicates its mitochondrial location