FIG. 2.
HIF-1α stabilization and ROS production during pharmacological inhibition of complex II. Wild-type Hep3B cells were treated with TTFA (200 μM) (A), 3-NPA (100 μM) (B), MMA (1 mM) (C), or combinations of these drugs (E) for 4 h under normoxic or hypoxic conditions. HIF-1α and β-actin were detected via immunoblotting. (D) Wild-type 143B cells were transfected with plasmids containing HRE-luciferase (HRE-Luc) and CMV-β-galactosidase and then treated with TTFA (200 μM), 3-NPA (100 μM), or MMA (1 mM) for 4 h. Lysates were analyzed for luciferase activity and corrected for transfection efficiency using β-galactosidase. *, P < 0.05. (F) 143B cells were infected with an adenovirus encoding roGFP, allowed to recover for 24 h, and imaged every 60 s during perfusion with buffer containing the indicated compounds for 30 min. The ratio of baseline oxidation of roGFP is indicated as 100%; values greater than 100% represent oxidation, whereas values less than 100% represent the reduction of the level of the sensor relative to baseline redox levels. (G) Superoxide production as assessed by DHE fluorescence in wild-type 143B cells treated with AA (1 μM), TTFA (200 μM), 3-NPA (100 μM), or MMA (1 mM). After treatment for 5 h, DHE (1 μM) was added for an additional hour. Cells were trypsinized, collected, and analyzed by flow cytometry. Values are shown as percentages relative to values for controls. *, P < 0.05. (H) Cells were treated with the indicated compounds for 5 h, after which Mito-SOX (1 μM) was added for an additional hour. Cells were trypsinized, collected, and analyzed by flow cytometry. Cont, control. Values are shown as percentages relative to values for controls. *, P < 0.05.