Figure 5

Human POT1 variants have important roles in maintaining telomere overhangs. (a) 293T cells transiently co-expressing GST-tagged TPP1 and Flag-tagged human POT1 variants were immunoprecipitated (IP) with anti-Flag antibodies and blotted as indicated. In addition to wild-type (wt) POT1 variant proteins, rescue constructs encoding POT1 variants that contained sgRNA-resistant silent mutations (rescue) were also examined. POT1 KO cells stably expressing the sgRNA-resistant rescue constructs were then induced with doxycycline for 6 days before being harvested for the assays described below. (b) For telomere ChIP analysis, the rescue cells were immunoprecipitated (IP) with anti-HA antibodies to bring down associated telomeric DNA for dot-blotting and hybridization with a telomere repeat probe (TTAGGG)₃ (left panel). IgG was used as a negative control. Telomeric signals were quantified and normalized against Alu repeat signals and graphed on the right. Error bars indicate s.e. P-values were obtained using one-way analysis of variance (ANOVA). *P<0.05, **P<0.01. (c) To assess RPA1 recruitment to telomeres, cells were immunostained with antibodies against RPA1 along with a telomere PNA probe. The data were quantified and graphed as shown. Three independent experiments were carried out for each cell line with at least 100 cells in each experiment. Error bars indicate s.e. (n=3). P-values were obtained using the Student’s t-test. **P<0.01, ***P<0.001. (d) To assess possible changes in TIFs, cells were immunostained using antibodies against 53BP1 along with a telomere PNA probe. The data were quantified and graphed as shown. Three independent experiments were carried out for each cell line with at least 100 cells 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<0.05, ***P<0.001. (e) Genomic DNA was extracted from the cells for processing in the presence (+) or absence (−) of Exonuclease I (ExoI) before hybridization analysis of ss G overhangs in the native gel using the (CCCTAA)₃ probe. Total telomeric DNA and genomic DNA (Alu) signals were determined under denaturing conditions. (f) Overhang signals from e were quantified and normalized against Alu repeat signals and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. (n=3). P-values were calculated using one-way analysis of variance (ANOVA).