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Fig. 7. Downregulation of SENP5 leads to increased reactive oxygen species (ROS) production. (A) COS-7 cells stably expressing either vector (top panels) or silenced for SENP5 shRNA (middle and bottom panels) were incubated at 37°C with 10 µM hET; oxidized hET (red) and MitofluorRed (green) were observed at the times indicated. Note the increased intensity of red fluorescence at 60 minutes in cells that contain shRNA targeting SENP5. The bottom panel shows a higher magnification of the aberrant mitochondria within these cells. (B) Average fluorescence intensity per cell of oxidized hET and MitofluorRed from 244 SENP5-silenced cells and 270 cells containing the vector. Data were collected (using identical laser intensities and scanning parameters) from live cells over a 90-minute period, imaging 10-20 cells per field. (C) Average fluorescence of oxidized hET per cell was plotted for each field as a function of imaging time. Each point represents the average hET fluorescence of 10-20 cells. Control cells did not significantly increase their ROS production over time, whereas the rate of ROS production in SENP5-silenced cells is increased. (D) COS-7 cells stably expressing either vector or shRNA targeting SENP5 were harvested and duplicate samples of 105 cells were incubated at 37°C with 10 µM hET for 40 minutes in culture medium. Then, fluorescence emission scans were collected to examine the reduced (emission peak, 420 nm; excitation, 355 nm) and oxidized hET (emission peak, 620 nm; excitation, 518 nm). Each value represents the mean ± s.d. of two independent aliquots (*P<0.01). (E) Same as D, but cells had been previously infected or not with DRP1-K38E-CFP-HA. Each value represents the mean ± s.d. of two independent aliquots (*P<0.01, **P<0.01).
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