Figure 3
Deletion of individual subunits impacts telomere end protection. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline for 6 days before being collected and used in the following assays. (a) The cells were examined by IF-fluorescence in situ hybridization (FISH) using antibodies against 53BP1 and the respective targeted proteins along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Scale bars 10 μm. (b) Data from a were quantified and graphed. Three experiments with independent doxycylcine inductions were carried out for each cell line, with at least 100 cells analyzed in each experiment. Only cells with at least five TIFs were counted as positive. Error bars indicate s.e. (n=3). P-values were obtained using the Student’s t-test. ***P<0.001. (c) The cells were harvested for metaphase spread and FISH analysis using a telomere probe. Representative images are shown here for cells induced to KO TIN2 and TRF2. White arrows indicate chromosome fusions. Scale bars 5 μm. (d) Data from c were quantified and graphed as indicated. At least 50 metaphases were scored for each sample. White arrowheads indicate chromosome fusions. Scale bars 5 μm. P-values were determined by one-way analysis of variance (ANOVA). (e) The cells were immunoblotted using the indicated antibodies. The anti-SMC1 antibody served as a loading control. γ-irradiated Hela cells (IR+) served as positive controls. (f) Data from e were quantified. At least three experiments with independent doxycycline inductions were performed and the results were combined. Signals for phosphorylated Chk1 and Chk2 were normalized against SMC1 signals and graphed as indicated. Error bars indicate s.e. P-values were obtained using the Student’s t-test. *P<0.05, **P<0.01.
