CPI-613 induces a strong mitochondrial burst of reactive oxygen species implicated in cell death. (A, B) Intracellular ROS levels were assayed using the hydrogen peroxide-sensitive dye DCF followed by FACS quantification. CPI-613 treatment causes a dose-dependent increase in whole cell DCF signal several-fold higher (A) than that caused by classical ROS inducing agents such as rotenone and TTFA at their conventional doses (B). Results are representative of three experiments. (***P <0.0005 compared to vehicle control; Student’s t test; n = 3). (C) Upper panel: Levels of dimerization (oxidization) of the cytosolic (Prx1) and mitochondrial (Prx3) isoforms of peroxiredoxin induced by CPI-613 or the mitochondrial inhibitor of ROS detoxification, auranofin, as a control were assayed by western blot (under oxidizing gel conditions). Lower panel: parallel samples were treated with 100 mM DTT and run under reducing gel conditions (converting all peroxiredoxin to the reduced monomer form and serving as a loading control). (D) CPI-613-induced Prx3 dimerization is dose dependent (left) and inhibited by co-treatment of cells with the antioxidant NAC (right). (E) NAC protects from cell death induced by CPI-613 as assayed by long-term ATP levels (16 hours) and cell morphology. Error bars represent SEM. DCF, 2',7'-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; NAC, N-acetylcysteine; TTFA, thenoyltrifluoroacetone.