Figure 6.

ROS induction by MitoVES correlates with CII inhibition in isolated mitochondria at low succinate. (a and b) Mitochondria isolated from variant cells respiring on 500 μM succinate in an oxygraph chamber were exposed to increasing concentrations of MitoVES, and ROS production was followed in real time in the presence of Amplex Ultra Red and peroxidase. (a) Shows reduced ROS production for I56F and S68A variants compared with WT, (b) shows the same for R72C and B9 cells. The same WT data are used in a, b, the panels are separated for clarity only. (c) Under the same conditions, 5 mM malonate inhibits ROS generation by 70 μM MitoVES in all cell lines except for B9 cells. (d) Respiratory data extracted from the experiments shown in a reveal that the respiration of WT cells is inhibited by MitoVES most efficiently. (e) ROS were measured as in a, b, but at 10 mM succinate. No increase in ROS generation was detected. (f) At 10 mM succinate there is no effect of 5 mM malonate on ROS at 70 μM MitoVES. Data represent the mean±S.E.M. of 3–5 independent experiments. Significant differences from WT: *I56F, **S68A, ^#B9, ^##R72C. Panel c: * denotes a significant decrease after the addition of malonate