Mitochondrial fragmentation is required for redox signaling during repair. (A) RhoA activity (RhoA-FLARE) in an injured WT MEF. (B) Plot showing mROS (MitoSOX) increase in response to PM injury in WT MEFs (n = 12 cells). (C) Lifeact-mCherry kinetics after PM injury in WT MEFs (n = 12 cells). (D) Images of MEFs costained with MitoTracker Green (left) and MitoSOX Red (right). MitoSOX images are shown before and 30 s after PM injury. (E) Basal mitochondrial ROS level in the absence of injury (n = 16 WT, 20 untreated DRP1 KO, and 14 rotenone-treated DRP1 KO MEFs). (F) Mitochondrial ROS increase in response to PM injury (n = 12 WT, 18 untreated DRP1 KO, and 11 rotenone treated DRP1 KO cells). (G) F-actin (Lifeact-mCherry) response to PM injury. Dotted box indicates injury-proximal region used for quantification. (H) Change in Lifeact-mCherry intensity after PM injury in WT (n = 12), DRP1 KO (n = 46), and rotenone-treated DRP1 KO MEFs (n = 37 cells). (I) Percentage of untreated and rotenone-treated DRP1 KO cells that failed to repair (n = 5 experiments). Arrows mark the site of injury. Scale bar = 5 µm (D) and 10 µm (A and G). *, P < 0.05 by unpaired t test. #, P < 0.05 by Mann–Whitney U test. Error bars represent SEM.