Fig. 1.

Mito-ROS production in D₅R-HEK293 cells. A Characterization of purified mitochondria (Band 3, as described in Supplementary Fig. 7) by immunoblotting with antibodies against markers for mitochondria (prohibitin), endoplasmic reticula (calnexin), Golgi bodies (GM130), nuclei (histone B4), and peroxisomes (catalase). TCL total cell lysate. B H₂O₂ production in isolated mitochondria, with pyruvate (5 mM) and malate (5 mM) as the substrates. Veh vehicle, Fen fenoldopam (D₁R/D₅R agonist, 1.0 μM, 12 h), Sch Sch23390 (D₁R/D₅R antagonist 1.0 μM, 12 h), AA antimycin A (Qi site inhibitor of mitochondrial ETC Complex III, 0.75 μM, 2 h), Cat catalase (10 μM, 30 min prior to AA treatment). Open rectangles, empty vector-transfected HEK293 (EV-HEK) cells; filled rectangles, D₅R-overexpressing HEK293 (D₅R-HEK) cells. n = 4/group, *P < 0.05 vs Veh, ^#P < 0.05 vs EV-HEK. C D₅-RHEK293 cells were treated with Veh or Fen (1.0 μM, 12 h) in the absence or presence of Sch (1.0 μM, 12 h). Mitochondria were stained with MitoTracker Green; mito-ROS were monitored with MitoSOX Red. Bar, 20 μm. The images are from one of three independent experiments. D The MitoSOX Red fluorescence density was obtained in 3–5 random fields from 25–40 D₅R-HEK293 cells in three independent experiments, as described in C; *P < 0.05 vs Veh