A. Immunoblot for total ATM, phosphorylated ATM, total CHK2 and phosphorylated CHK2 in hNBS-ILB1 cells expressing indicated proteins. γ-tubulin: loading control. B.
hNBS–ILB1 cells expressing the indicated DNAs were exposed to TRF2ΔBΔM and telomeres were visualized by PNA-FISH (red), anti-53BP1 antibody (green) and DAPI (blue). Arrowheads point to 53BP1 positive TIFs. C. Quantification of percentage of cells containing ≥ 5 53BP1 positive TIFs in (B). Data represents the mean of two independent experiments ± SEM; n>100 nuclei analyzed per experiment. **: p<0.003, ***: p<0.0007, one-way Anova. NS, non-significant. D.
hNBS–ILB1 cells (top) or Nbs1−/− MEFs (bottom) expressing either WT NBS1 or NBS1 serine mutants were infected with either control vector, TRF2ΔBΔM or shTrf2. FITC-OO-(TTAGGG)4 (green, leading strand), Tam-OO-(CCCTAA)4 (red, lagging strand), DAPI (blue) were used to visualize fused chromosomes (arrowheads). E. Quantification of telomere fusion frequencies in (D). Data represents the average of three independent experiments as mean ± SEM from a minimum of 70 metaphases. ***: p<0.0002, ****: p<0.0001; one-way Anova. See also Figure S5.