Figure 5.

Mitochondrial targeting of survivin in INS-1 cells inhibits apoptosis. (A) Cytoprotection in stable transfectants. Parental INS-1 cells or INS-1 cells stably transfected with Surv, MT-GFP, or MT-S were treated with staurosporine and analyzed for caspase-3 and caspase-7 activity (DEVDase activity, green fluorescence) and plasma membrane integrity (propidium iodide, red fluorescence), by multiparametric flow cytometry. The percentage of cells in each quadrant is indicated. (B) Caspase-3 cleavage. INS-1 cells expressing MT-GFP or MT-S were treated with staurosporine and analyzed at the indicated time intervals by immunoblotting. The position of active caspase-3 bands of 17 and 19 kDa is shown. (C) Caspase-9 cleavage. The experimental conditions were as in B except that samples were analyzed for generation of 37-kDa active caspase-9 fragments by immunoblotting. (D) Inhibition of caspase-dependent apoptosis. Parental INS-1 cells or INS-1 cells stably expressing MT-GFP or MT-S were treated with the indicated concentrations of staurosporine in the presence or absence of zVAD-fmk, and scored for nuclear morphology of apoptosis by DAPI staining. Data are the mean ± SEM of 3 independent experiments.