Figure 4. NBS1^S432 phosphorylation is cell cycle regulated and required to protect newly replicated telomeres.

A. Fucci U2OS cells transfected with indicated DNA constructs were FACS sorted to purify G1 and S/G2 cells. Sorted cells were immunoprecipated with anti-Myc antibody and then immunoblotted with anti-Myc and anti-Flag antibodies. Inputs represent 5% of the total cell lysate used for the IP. Cyclin A was used to mark S/G2 phase of the cell cycle. B. U2OS cells synchronized with 2mM thymidine and 1.0 μg/ml aphidicolin were fixed, telomeres visualized by PNA-FISH (red), NBS1 visualized by anti-Flag antibody (green), and DAPI (blue). Anti-cyclin A antibody was used to mark S/G2. C. Metaphases prepared from Nbs1^−/− MEFs reconstituted with WT mNBS1, mNBS1^S433 mutants or PNUTS^ΔC were analyzed by CO-FISH. FITC-OO-(TTAGGG)4 (green, leading strand), Tam-OO-(CCCTAA)₄ (red, lagging strand) and DAPI for chromosomes (blue). Arrowheads indicate leading-leading chromatid fusions. D. Quantification of chromatid fusions observed in (C). E. Localization of Apollo/SNM1B in U2OS cells expressing WT Flag-mNBS1 or Flag-mNBS1^S433 mutants. Telomeres were visualized with PNA-FISH (red), anti-HA antibody to visualize Apollo/SNM1B (green), and DAPI (blue). F. Quantification of Apollo/SNM1B foci in (E). Data represents the mean of three independent experiments ± SEM; n>200 nuclei scored per experiment. *: p<0.01; one-way Anova. See also Figures S3 and S4.