Actin becomes stably associated with ETC-inhibited mitochondria. (A). Live-cell imaging of mitochondrial actin enrichment in fibroblasts treated for 1 h with 40 nM AntA or vehicle. Fibroblasts were cultured in galactose media to increase their dependence on ETC function. F-actin and mitochondria were visualized using GFP-F-tractin (green in merged image) and mito-mRaspberry (magenta in merged image). (B) Quantification of images treated as in A. n = 11–15 cells per condition from 4 independent animals. (C) Fibroblasts were transduced and treated with AntA as in A. Cells were imaged live on an Airyscan2 microscope. F-actin is shown as green, and mitochondria are magenta. (D) Fibroblasts transduced with GFP-Ftractin and mito-mRaspberry and cultured in galactose were treated with 40 nM AntA or vehicle as in A. One hour after drug treatment, cells either had glucose or galactose added to a concentration of 25 mM, or no further addition. Cells were imaged after 30 min. n = 10–11 cells per condition from 5 independent animals. For the top two rows of A, the scale bars are set to 20 μm; the bottom row of the same figure (enlargement) has scale bars set to 5 µm. The scale bar in C is 3 μm. The bars on boxplots show the 10–90th percentile. P values for B were calculated with two-tailed, unpaired t tests with Welch’s correction. For D, P values were calculated by performing a blocked one-way ANOVA with Tukey’s multiple comparison correction. Select P values are shown.