Mitochondrial ROS modulation by ClpXP.

<p>(<b>A</b> and <b>B</b>) PC3 cells transfected with control siRNA (Ctrl) or ClpX- or ClpP-directed siRNA were labeled with CellROX Green Reagent by flow cytometry (<b>A</b>), and staining intensity was quantified (<b>B</b>). H₂O₂ was a control oxidative stimulus. **, <i>p</i> = 0.004; ***, <i>p</i> < 0.0001. (<b>C</b>) The indicated prostate cancer cell types were analyzed for total ROS production as in (<b>A</b>). *, <i>p</i> = 0.03–0.04; **, <i>p</i> = 0.001; ***, <i>p</i> = 0.0008–<0.0001. (<b>D</b>) PC3 cells transfected with the indicated siRNAs as in (<b>A</b>) were analyzed for MitoSOX red mitochondrial superoxide reactivity by fluorescence microscopy and quantified. H₂O₂ was a control oxidative stimulus. **, <i>p</i> = 0.001; ***, <i>p</i> = 0.0004–0.0001. (<b>E</b> and <b>F</b>) PC3 cells stably transduced with control pLKO or shRNA to ClpX or ClpP were analyzed for mitochondrial superoxide production by fluorescence microscopy (<b>E</b>), and staining intensity was quantified (<b>F</b>). ***, <i>p</i> = 0.0004–0.0001. (<b>G</b>) PC3 cells stably transduced with pLKO or ClpX- or ClpP-directed shRNA were analyzed by western blotting. (<b>H</b>) PC3 cells transfected with control siRNA or ClpX- or ClpP-directed siRNA were incubated with vehicle (Veh) or the combination of antioxidants NAC (N) plus mitochondrial-directed MitoTempo (MT) and analyzed by western blotting in the absence or presence of the autophagic flux inhibitor hydroxychloroquine (HCQ). (<b>I</b>) Densitometric quantification of LC3-II/I ratio in vehicle (Veh) or antioxidant-treated PC3 cells in (<b>H</b>). Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002507#pbio.1002507.s003" target="_blank">S3 Data</a>.