Figure 4
Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. (a, b) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a 32P-labeled telomere probe (TTAGGG)3. Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a. Representative gels of the TRF assay are shown in b. (c) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. (d) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of Exonuclease I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the 32P-labeled (CCCTAA)3 probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. (e) Overhang signals for each cell line from d were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples 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 obtained using the Student’s t-test. **P<0.01.
