Figure 3

Elucidation of the ROS mediated, mitochondrial death pathway upon caspase activation. A, Expression of various apoptotic proteins following treatment of cells with complex 3 for 24 h, with β-Actin as loading control. B, Densitometric analysis of proteins detected by Western blot. C, Determination of caspase 9 and 3 activities at 405 nm, following treatment of cells with complex 3 for 24 h. D, Determination of caspase 9 and 3 activities at 405 nm in cells, incubated 1 h with Z-LEHD-FMK and Z-DEVD-FMK prior to treatment with complex 3 for 24 h. E, Percentage of growth inhibition of cells following incubation with caspase inhibitors Z-LEHD-CHO and Z-DEVD-CHO (for caspase 9 and caspase 3 respectively), along with complex 3 for 24 h. F, Cytochrome c release into the cytosol after treatment of cells with complex 3 as determined at 450 nm assay. G, Loss of ∆ψm of complex 3 treated cells after 24 h. H, Flowcytometric analysis of loss of ∆ψm in presence of complex 3 after 24 h. I, ROS generation upon treatment of complex 3 for 6, 12 and 24 h at 529 nm. J, ROS generated in cells treated with complex 3 and with varying concentrations of NAC (0.2, 1, 5 and 10 μM) after 24 h at 529 nm. K, Percentage growth inhibition of cells in presence or absence of either complex 3 or NAC or in presence of both of them. L, Flowcytometric analysis ROS generation in cells following treatment with complex 3 along with NAC after 24 h. Values are mean ± S.D and represent one of the 3 representative experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.