Figure 5
Suppression of CII-driven respiration correlates with cell death for Qp site inhibitors that do not rapidly increase the succinate level. (a) Digitonin-permeabilized respiration-competent variant cell lines respiring on 10 mM succinate in the presence of 0.5 μM rotenone and FCCP were exposed to increasing concentrations of MitoVES. The variants show reduced inhibition compared with WT. (b) Inhibition by the dicarboxylate-binding site inhibitor malonate in the same experimental set-up shows similar efficiency for all Qp site substitutions. (c) Similar to a and b, but the Qp site inhibitor TTFA was used. (a–c) The data represent the mean±S.E.M. of 3–4 independent experiments. (d) Variant cell lines were exposed to 0.5 mM TTFA for 24 h and the percentage of annexin V-positive cells was determined by flow cytometry. n=5, mean±S.E.M., * values significantly different from WT. (e) Cells were exposed to 250 μM TTFA for 30 min and the level of ROS was determined by dihydroethidium (DHE) staining and flow cytometry. n=5, mean±S.E.M., * values significantly different from WT. (f) Cells were transfected with catalase-coding or control vector, exposed to 2 mM TTFA for 20 h, and the percentage of annexin V-positive cells was determined by flow cytometry. n=4, mean±S.E.M., * values significantly different between catalase and mock-transfected cells. Inset, catalase overexpression verified by western blot. (g) Cells were exposed to 250 μM TTFA for 30 min in the presence or absence of 50 mM malonate (30 min pretreatment), and the level of ROS was determined by DHE staining and flow cytometry. n=4, mean±S.E.M., * values significantly different in the presence and absence of malonate. (h) Cells were exposed to 1.5 mM TTFA for 12 h in the presence or absence of 20 mM malonate (30 min pretreatment). The percentage of annexin V-positive cells was determined by flow cytometry. n=4, mean±S.E.M., * values significantly different in the presence and absence of malonate. (i) Atpenin A5-induced inhibition of respiration of permeabilized cells as described in a. n≥3, mean±S.E.M. (j) Cells were exposed to 1 μM Atpenin A5 for 24 h and the percentage of annexin V-positive cells was determined by flow cytometry. n=3, mean±S.E.M. (k) Cells were exposed to 0.5 μM Atpenin A5 for 30 min and the level of ROS was determined by DHE staining and flow cytometry. n=5, mean±S.E.M. (l) Succinate levels were determined in WT cells exposed to 20 μM MitoVES, 1 mM TTFA or 1 μM Atpenin A5 for 30 min. n=3, mean±S.E.M., * values significantly different from control
