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. 2017 Jul 24;40(7):503–514. doi: 10.14348/molcells.2017.0081

Fig. 2. PINK1/Parkin-independent changes in ROS levels by NAM treatment.

Fig. 2

Levels of mitochondria content (A) and superoxide (B) were determined using NAO or DHE at Day 1 or 2 of NAM treatment, and the effects were compared between normal fibroblasts and those whose Parkin expression was knocked down by siRNA (siParkin). The image of the protein blot (top) shows diminished Parkin protein levels in cells treated with siParkin RNA. Three independent biological samples (different transfections) were analyzed, and the averages normalized by those of untreated cells (wt) were plotted. *P < 0.1, **P < 0.05 (compared to wt control, oneway ANOVA). (C) Ubiquitin puncta formation was compared between cells treated with 10 μM CCCP for 4 h, or 5 mM NAM for 12 h. Fibroblasts were transfected with GFP-tagged human UBC gene 24 h earlier, and were treated with either CCCP or NAM, fixed, and then probed with OXPHOS antibodies to visualize mitochondria (blue). Cells positive for green fluorescence from ubiquitin-tagged GFP were photographed.