Figure 3

CPI-613-induced mitochondrial reactive oxygen species originate from a non-electron transport chain source. (A) ρ° cells lacking mtDNA-encoded components of the ETC were generated from H460 (ρ⁺) as described previously[26] and validated by western blot, demonstrating the absence of the mtDNA-encoded protein cytochrome c oxidase subunit 1 (COX1) but containing nuclear-encoded mitochondrial protein dihydrolipoamide dehydrogenase (E3). The nuclear-encoded cytosolic actin protein served as a loading control. (B) ROS levels were quantified following treatment with 240 μM CPI-613 or 4 μM antimycin-A using the superoxide-detecting dye DHE followed by FACS analysis. CPI-613 induced comparable amounts of ROS in ρ° and ρ⁺ cells whereas the complex III ROS inducer antimycin-A failed to increase DHE fluorescence in ρ° cells. Results are representative of three experiments. (C) ρ° H460 cells were assayed for oxidation of the mitochondrial Prx3 protein comparably to ρ⁺ in Figure 2C. These ρ° cells produced high levels of mitochondrial ROS by this assay despite lacking key ETC components. (D) The antioxidant NAC protects from CPI-613-induced cell death in ρ° cells as assayed by whole cell ATP levels after 20 hours of drug treatment. Error bars represent SEM. DHE, dihydroethidium; DMSO, dimethyl sulfoxide; mtDNA, mitochondrial DNA; NAC, N-acetylcysteine.