Figure 2

H₂O₂ induces mitochondrial apoptosis in COX-deficient cells. (a) HeLa cells pretreated with KCN (1 mM) for 24 h, 143B_ΔCOX cybrid cells and COX10^−/− fibroblasts were subjected to H₂O₂ in the absence (gray column) or presence of pan-caspase inhibitor zVAD-fmk (20 μM), caspase-8 inhibitor zIETD-fmk (2 μM) or caspase-9 inhibitor zLEHD-fmk (2 μM). Cell death was measured by trypan-blue exclusion after 20 h. (b) Cytchrome c and caspase-9 were detected in the cytosol of cells by WB after treatment with H₂O₂ for 6 h (HeLa), 12 h (143B_ΔCOX cybrid cells) or 16 h (COX10^−/−), respectively. The arrowheads indicate caspase-9 cleavage bands p35 and p37. Activated Bax was immunoprecipitated by 6A7 anti-Bax antibody in total lysates of HeLa cells and detected by WB after 4 h. (c) Wild-type (wt) and Bax/Bak double-knockout MEFs (Bax^−/−Bak^−/−) were pretreated with KCN (200 μM) for 24 h or left untreated. Cell death upon treatment with H₂O₂ was measured after 18 h by trypan-blue exclusion. The release of cytochrome c was detected by WB after 6 h. (d) HeLa and HeLa Bcl-2 overexpressing cells were pretreated with KCN (1 mM) for 24 h or left untreated. Cell death upon treatment with H₂O₂ was measured after 18 h by trypan-blue exclusion and release of cytochrome c was detected by WB after 6 h. The error bars in panel a represent the mean±S.D. (n=4), the error bars in panel c and d represent the mean±S.D. (n=3), two-tailed unpaired t-test. *P<0.05, **P <0.01, ***P<0.001