Oligomycin-induced ADA in MEFs. (A) Mitochondrial polarization (assessed by TMRE fluorescence) in MEFs with DMSO, 1 µM CCCP or 1.5 µM oligomycin (± SEM) treatment. n ≥ 118 cells per group combined from two independent experiments. Experiments done in 2 mM glucose without serum. (B) MEFs stained for actin filaments (TRITC-phalloidin, green), mitochondria (Tom20, red) and DNA (DAPI, blue) after 5 min treatment with DMSO, 1.5 µM oligomycin or 1.5 µM oligomycin with 100 µM CK666. Bottom images are zooms of boxed regions. Experiments done in 2 mM glucose without serum. Scale bars: 10 and 5 μm. Arrow indicates actin assembly. (C) % cells (± SEM) displaying ADA for the conditions shown in panel B. n ≥ 65/14 cells/fields of view (FOV) per group combined from two independent experiments. **** P < 0.0001. Statistical significance was calculated using unpaired two-tailed t tests. Experiments done in 2 mM glucose without serum. (D) Graph of actin intensity (± SEM) around mitochondria in MEF cells as a function of time for 1 µM CCCP or 100 µM CK666 + 1 µM CCCP simultaneous treatment. Cells were cultured in Agilent seahorse DMEM supplemented with 1 mM glucose and 4 mM glutamine but without serum for 1 h before imaging. n ≥ 35 cells per condition combined from two independent experiments. Arrow indicates time of treatment. **** P < 0.0001. Statistical significance at indicated timepoint was calculated using unpaired two-tailed t tests. Number of experiments, statistical tests, and sample sizes are provided in Table S1.