Fhl2, Myo6, and PINK1 are not implicated in mitochondrial actin association after ETC inhibition. (A) Anti-FLAG immunolocalization of FLAG-Fhl2 (magenta in merged image) in fibroblasts treated for 1 h with vehicle or 4 nM AntA. The FLAG-Fhl2 construct also expressed a P2A ribosome skip site followed by a mitochondria-targeted GFP (green in the merged image) to visualize mitochondria. (B) Quantification of images treated as in A. n = 10–12 cells per condition from 3 independent transductions. (C) Quantification of mitochondrial movement in cultured hippocampal neurons expressing mito-dsRED and meGFP and nontargeting control shRNA or a shRNA targeting Fhl2. Three days after transfection, cells were treated for 1 h with either 4 nM AntA or vehicle and then imaged. n = 7–9 axons per treatment from three independent animals. (D) Fluorescence microscopy of fibroblasts expressing GFP-Myo6 (green in merged image) and mito-mRaspberry to mark mitochondria (magenta in merged image). Cells were treated for 1 h with vehicle, 4 nM AntA, or 20 µM CCCP. (E) Quantification of images treated as in C. n = 10–23 cells per condition from 3 independent transductions. (F) Quantification of perimitochondrial actin enrichment comparing vehicle and AntA-treated wild-type and PINK1 knockout fibroblasts treated for 1 h with vehicle or 40 nM AntA. n = 10–12 cells from 4 independent transductions. The P value for B was calculated by a two-tailed, unpaired t test with Welch’s correction. An ANOVA with Dunnett’s T3 multiple comparison correction was performed for E. Blocked one-way ANOVAs were performed with Tukey’s multiple comparison correction for C and F. Select statistical comparisons are shown.