Figure 2. RR and ATM are required for increased mtDNA copy number and biogenesis in response to IR and proper regulation of RR subunit expression.

(A) Relative mtDNA levels (plotted as in Figure 1) in primary wild-type fibroblasts 24 and 48 hours after exposure to 17.5 Gy of IR with all values compared with nonirradiated, untreated wild-type cells at 24 hours. Cells were exposed to 1 μM Triapine for 24 hours either immediately after IR (24 hours) or after a 24-hour recovery (48 hours) as indicated. The mean ± SEM is plotted. A 1-way ANOVA was used to determine statistical significance as indicated. (B) Relative mtDNA copy number of wild-type and A-T primary fibroblasts 24 and 48 hours after exposure (+) to 17.5 Gy of IR is plotted as in Figure 1. (C) Mitochondrial mass of wild-type and A-T patient fibroblasts 48 hours after IR. Median MitoTracker Green FM fluorescence intensity (mean ± SEM) is plotted. FACS histograms from 1 representative replicate are shown in the right panels. The percentage of cells collected (% max; y axis) with the indicated amount of fluorescence on the x axis (log scale) is shown. (D) Western blot analysis of R1, R2, and p53R2 (p2) from nonirradiated A-T cells (C) or WT and A-T cells 48 hours after 17.5 Gy (IR) from a representative experiment. Actin was probed as a control (C) for protein loading (the relative amount of actin in each lane is shown below the actin panel; wild-type nonirradiated was set to 100%). Actin-normalized signals from 3 independent experiments are depicted graphically on the right of the panels with the wild-type nonirradiated control protein levels set to 1 as indicated by the dotted line.