Fig. 3.

Recovery of mtDNA copy number following mtDNA depletion recruits Rad51 to the mitochondria. (A) U20S cells were treated for 2 h with 10 μg/mL BrdU, then stained with MitoTracker Deep Red and anti-BrdU (green). Yellow/green spots embedded in mitochondria indicate sites of nascent mtDNA synthesis. (B) Inhibition of mtDNA labeling was achieved with a 2 h pre-treatment with 50 ng/mL EtBr prior to the BrdU pulse. (C) U20S cells were treated with either 20 μM ddC or 50 ng/mL EtBr for 2 days, then washed and allowed to recover for 4 days. Total DNA was isolated every 24 h for qPCR determination of copy number per cell with changes being calculated relative to unstressed controls and normalized to the 18S rRNA gene. Fold change in total mtDNA in each population was determined by multiplying copy number per cell by relative cell count for each day. Error bars indicate S.E. (n = 4). (D) U20S cells were transfected with either nonsilencing or Rad51-specific siRNAs. Both populations of cells were treated with 50 ng/mL EtBr for 2 days, then washed and allowed to recover for 4 days. Total DNA was isolated every 24 h for qPCR determination of mtDNA copy number per cell. Error bars indicate S.E. (n = 4). (E) Western blot of Rad51 in purified mitochondrial fractions isolated from cells not treated with polymerase inhibitors (untreated, Unt), cells treated with either inhibitor during the first 24 h of the depletion phase (Dep) and cells 24 h post drug removal (Rec, day 3 in panels C and D). ATP synthase serves as the loading control. (F) Western blot analysis of Rad51 in total cell lysates that were prepared every 24 h during the depletion and recovery phases. GAPDH serves as the loading control. All blots are representative of 4 independent experiments.