Figure 5
Loss of caspase-2 protects cells from mitochondrial oxidant-induced cell death. (a) Primary skin fibroblasts from newborn WT or Casp2−/− mice on glass bottom chambers were treated with stressors as indicated (STS= 0.5 μM staurosporine for 6 h; ROT=0.5 μM rotenone for 30 h), stained with propidium iodide (PI) and Hoechst and imaged on a widefield microscope; representative photomicrographs are shown. (b) Percent PI-positive cells after normalization to total number of cells (Hoechst positive), followed by ratio to cell death in untreated cells. (c) Primary skin fibroblasts from newborn WT or Casp2−/− mice were seeded in black bottom 96-well plates and treated with stressors as indicated (STS= 0.5 μM staurosporine for 15 h; ETO=100 μM etoposide for 24 h; ROT= 0.5 μM rotenone for 30 h; AA= 10 μM antimycin A for 24 h; OLG= 23 μM oligomycin for 15 h). After treatment, cells were stained with PI and cell death levels were quantitated by measuring fluorescence on a plate reader. Digitonin was then added to lyse all cells and plates were read again. Percent cell death was calculated as the percent ratio of fluorescence intensities before and after digitonin treatment. Data represent values from three independent experiments. Data were analyzed using two-way ANOVA and Bonferroni post-tests.
