(A) U2OS cells overexpressing Spire1C display shorter mitochondria (second panel, 2.2 ± 0.5 μm, nmitochondria = 211, ncells = 14, p < 0.0001), whereas cells overexpressing Spire1C mWH2 (third panel, 6.2 ± 1.52 μm, nmitochondria = 332, ncells = 15, p < 0.0001) or Spire1CΔKIND (fourth panel, 9.0 ± 1.50 μm, nmitochondria = 232, ncells = 16, p < 0.0001) display longer, more tubulated mitochondria compared to control cells (first panel, 3.57 ± 0.45 μm, nmitochondria = 322, ncells = 34). Scale bar: 10 μm. (B) Cells transfected with mitoEmerald (and neighboring non-transfected cells) stained with α-ExonC (upper row) showed robust colocalization of mitoEmerald and α-ExonC, with a mixture of tubulated and fragmented mitochondria. Cells cotransfected with Spire1 shRNA and mitoEmerald with no detectable α-ExonC labeling (lower row) display long, tubulated mitochondria (6.1 ± 1.26 μm, nmitochondria = 222, ncells = 17). All primary antibodies were counterstained with Alexa-568 secondary antibody. Scale bar: 15 μm. (C) Left: Average mitochondrial lengths for control cells and cells overexpressing Spire1C, Spire1C mWH2, Spire1 shRNA or Spire1CΔKIND. Right: Average number of mitochondrial fission events in one cell in a timespan of 10 min for control (ncells = 17), Spire1C overexpressing (ncells = 10, p < 0.0001), Spire1C mWH2 overexpressing (ncells = 12, p < 0.0001), Spire1 knockdown (ncells = 25, p < 0.0001) and Spire1CΔKIND overexpressing (ncells = 22, p < 0.0001) cells. At least 3 separate experiments were performed for all conditions. Error bars represent standard error of the mean.