Figure 4.
Effects of Mito-HNK on Mitochondrial Complex I Activity, ROS, and the Redox State of Peroxiredoxins
To measure complex I activity, cells were pretreated for 24 hr with Mito-HNK and HNK, the cell membrane was permeabilized, and OCR was measured upon the addition of mitochondrial substrates/inhibitors.
(A and B) Both HNK (IC50 = 30 μM for both cell lines) and Mito-HNK (IC50 = 0.1 μM for both cell lines) inhibit complex I in H2030-BrM3 NSCLC cells (A) and DMS-273 SCLC cells (B).
(C and D) The effect of Mito-HNK (1 μM, 24 hr treatment) on cellular ROS production, as measured by HPLC-based profiling of the oxidation products of the HE probe in H2030-BrM3 (C) and DMS-273 (D) cells. The compound 2-hydroxyethidium (2-OH-E+) is a specific product for superoxide and diethidium (E+-E+) is a marker product for one-electron oxidants. HPLC traces are shown in the left panels and the quantitative results in the right panels (**p < 0.01, ***p < 0.001).
(E and F) The 24-hr treatment of H2030-BrM3 cells with 0.2 μM Mito-HNK (E) or DMS-273 cells with 0.3 μM Mito-HNK (F) leads to significant oxidation of mitochondrial Prx3, whereas the oxidation state of cytosolic Prx1 is not significantly affected (**P < 0.01, ***P < 0.001 vs.control). Error bars indicate SD.