Actinonin induces a loss of mitochondrial membrane potential and activation of Oma1. (A) Immunoblot of wild-type and KO Oma1 MEFs treated with actinonin or ethanol (−) for the indicated time. (B) Quantification of mitochondrial membrane potential with TMRM staining in HEK293 cells treated for 6 h with chloramphenicol (CA) and actinonin (Act). Ethanol-treated cells were used as the control, and CCCP was used as a positive control to dissipate the membrane potential. The data represent four independent experiments. (C) Representative confocal images of mitochondrial morphology from Oma1 wild-type or KO MEFs treated with ethanol or actinonin for 6 h. The arrows indicate bulbous mitochondrial morphology. Bars, 10 µm. (D) Quantification of mitochondrial morphology from Oma1 wild-type or KO MEFs treated with ethanol (Etoh) or actinonin for 6 h. Data from three independent experiments with 100 cells counted per experiment. B, bulbous; F, fragmented; I, intermediate (mix of fragmented and tubular); T, tubular.