Akt inhibition induces mitochondrial superoxide and cellular ROS production, which is augmented by CQ. (A) PC3 cells cultured in 0.5% FBS were treated with DMSO, 3 μM Akti-1/2, 10 μM CQ, or both, stained with MitoSOX red dye, and examined by fluorescence microscopy. Images at 24 h are shown. Bar, 10 μm. (B) PC3 cells treated as in A were stained with the Image-iT LIVE green ROS Detection kit and examined by fluorescence microscopy at 24 h. Bright field (BF) images of cells are also shown. Bar, 10 μm. (C) Quantification of MitoSOX red and ROS green fluorescence intensities by flow cytometry at 24 h. Cells were treated as in A and B. Error bars represent SEM (n = 3).