Figure 8. Neuronal cell death occurs by triggering mitochondrial-dependent apoptotic pathway in Hebp1-expressing neurons.

Western blot analyses of (A and B) Cyt_C and Mic60 leakages and (C) caspase 9 activation in Hebp1-deficient, wildtype and control neurons. Wildtype and control neurons exhibited high levels of activated caspase 9 concomitant with mitochondrial release of Cyt_C and Mic60 into the cytosol (S). Hebp1 release was also coupled with leakage of Cyt_C and Mic60 in these cells. In contrast, Hebp1-deficient neurons displayed no apparent activation of caspase 9 despite leakages of Cyt_C and Mic60 from neuronal mitochondria (Mt). (D) Wildtype, control and Hebp1-deficient neurons were treated with 10 µM hemin for 24 hr. Apoptotic cells were visualized by fluorescence staining corresponding to caspase 3/7 activation (see Materials and methods). Hebp1-deficient neurons demonstrated resistance to apoptosis upon heme overload, whereas wildtype and control neurons exhibited high levels of caspase 3/7 activity. (E) Quantification of the data represented by the images shown in (D). All bar charts represent mean ± SEM. Statistical significance in the datasets was assessed by one-way ANOVA followed by Student’s t-test comparison for individual pairs of samples: ****p<0.001. All data shown are representative of results obtained from three independent experiments.