mtDNA damage results in mitochondrial dysfunction. (A) Representative images of cells expressing Cox4::GFP stained with TMRE after treatment with FCCP (2 μM) for 20 min or Oligomycin (0.5 μg/ml) for 4 h (left panel). Total TMRE intensity normalized to cell area is plotted; n > 650 pooled from three biological replicates for FCCP and Oligomycin and two biological replicates for empty vector (right panel). (B) Oxygen consumption rate (OCR) estimated using Seahorse assay after 4 h of induction. Mean and SD is plotted from three biological replicates. (C) Cox2 Western blot with quantification from three biological replicates, normalized to empty vector at t = 0; error bars: SD. (D) Representative images of cells expressing Cox4::GFP stained with TMRE after 4 h of induction with 100 nM β-estradiol. Examples show cells from three different morphological categories for both empty vector and mtDarT (left panel). Total TMRE intensity normalized to cell area is plotted for cells with three mitochondrial morphologies, after 4 h of induction (for empty vector, n = 215 for tubular, n = 35 for fragmented, n = 30 aggregate; for mtDarT, n = 258 for tubular, n = 79 for fragmented, n = 118 for aggregate; right panel). Cells were pooled from three biological replicates. Statistical significance in A and D was calculated using Mann–Whitney U test. Source data are available for this figure: SourceData FS4.