ADA in multiple cell types. (A) Micrographs of live-cell imaging for U2-OS, HeLa, Cos-7, and MEF cell before (0 s) and at their peak ADA timepoints after 20 µM CCCP addition (150 s–U2-OS; 135 s -HeLa; 210 s–Cos-7; 225 s–MEF). All cells were transfected with markers for actin filaments (GFP-F-tractin, green) and mitochondria (mito-DsRed, red). Scale bars are 10 μm (full cell) and 5 μm (inset). Yellow arrow indicates punctate mitochondrion without actin assembly. Corresponds to Videos 1, 2, 3, and 4. (B) Graph of actin intensity (± SEM) around mitochondria in U2-OS, HeLa, Cos-7, and MEF cells as a function of time of 20 µM CCCP or 100 µM CK666 + 20 µM CCCP simultaneous treatment. Cells per condition: U2-OS, n = 31; HeLa, n = 32; Cos-7, n ≥ 17; MEF, n ≥ 22 combined from two independent experiments. Arrows indicate time of treatment. **** P < 0.0001. Statistical significance was calculated between the indicated timepoint using unpaired two-tailed t tests. Experiments done in 25 mM glucose with serum. Exact number of experiments, statistical tests, and sample sizes are provided in Table S1.