Fig. 5.

ROS levels fluctuate during prolonged exposure to HG conditions. (A) ROS levels were measured in Clone 9 cells with DHE every 2 h after incubation with 50 mM glucose. Fluorescence intensity was normalized against the intensity at time 0 (F/F₀). The second phase of ROS increase was observed at 10-h incubation, and the ROS levels remained high until the 18-h time point, then decreased thereafter. (B and C) Fragmented mitochondria were prevalent in cells incubated for 10–18 h, and mitochondria returned to tubular morphology at 22–24 h in HG (gray bars in C). Mitochondria from cells incubated for 16 and 22 h are shown in B. Cells overexpressing DLP1-K38A maintained elongated tubular mitochondria during prolonged incubation with HG concentrations (C, solid bars). (D) Cells overexpressing GFP-DLP1-K38A (arrows in D) showed little change in ROS levels, which remained low throughout the extended incubation in HG (black bars) whereas ROS levels in untransfected cells (gray bars) increased and decreased.