Figure 6. SIRT3 controls intracellular ROS levels.
A, C2C12 myotubes were infected with adenovirus expressing GFP or SIRT3. DHE (5 µM) was added to media 30 min before collecting cells. The cells were harvested and immediately analyzed with a flow cytometry, and data were processed with Expo 32ADC software. The histogram overlays showed overexpression of mSIRT3 reduced the cellular ROS level. B, Knockdown of PGC-1α or SIRT3 increased cellular ROS level. C2C12 myotubes were treated with the indicated adenovirus (Ad-siControl, Ad-siPGC-1α or Ad-siSIRT3), treated with DHE, and analyzed with a flow cytometry as described in panel A. C, Overexpression of PGC-1α decreased cellular ROS level, and knockdown of SIRT3 blocked the inhibitory effect of PGC-1α on ROS production. C2C12 myotubes were treated with the indicated adenovirus (Ad-GFP, Ad-PGC-1α, Ad-siControl, or/and Ad-siSIRT3), treated with DHE, and analyzed with a flow cytometer as described in panel A. x-Axis, fluorescence intensity showing the extent of DHE oxidation; y-axis, cell number. The image is representative of three experiments. Right panels, Flow cytometry analysis of myotube cells. Values represent the mean of four independent experiments performed in duplicate. MFI, mean fluorescent intensity. *, P<0.05.