Figure 2.

MDC1 stimulates NHEJ of dysfunctional telomeres. (A) Metaphase spreads from TRF2F/− p53^−/− and TRF2F/+ p53^−/− MEFs expressing control luciferase or mouse MDC1-specific shRNA, sh4, 60 h after Cre infection. Telomeric signals were detected with FITC-OO-(AATCCC)₃ oligonucleotide and are false-colored in green; DNA (DAPI) is false-colored in red. (B) Quantification of telomere fusions in TRF2F/− p53^−/− MEFs, expressing the indicated shRNA, mock- or Cre-infected for 60–72 h. Telomere fusions were counted as events per chromosome in metaphase spread as shown in A. Bar graph represents fusion frequency relative to control shRNA-treated cells in three independent experiments. (C) Telomeric DNA analysis. (Left) In-gel assay detecting 3′ overhang of TRF2F/− p53^−/− MEFs expressing control luciferase or MDC1 shRNAs, sh4 and sh5. Cells were harvested 72 h post-infection with Cre and processed by in-gel hybridization to a (CCCTAA)₄ probe to detect ssTTAGGG repeats (native). (Right) The DNA was denatured in situ and rehybridized to the same probe to detect the total TTAGGG signal (denatured). Overhang signals were quantified with ImageQuant software and normalized to the total TTAGGG signal in the same lane. The numbers below the gel represent the percentage of normalized overhang signal compared with the normalized overhang signal for the same cells not treated with Cre. (D) Quantification of telomeric overhang 72, 96, and 120 h post-infection with Cre in two independent experiments. The overhang signal at different time points after Cre infection is represented as a percentage of the overhang signal in the absence of Cre for the same cell line.