Figure 7. Telomere replication is profoundly delayed when Timeless is depleted. (A) Timeless depletion caused a profound delay in telomere replication timing. HeLa cells expressing control or Timeless shRNA were synchronized by thymidine at the beginning of S-phase and released. Cells were incubated with EdU for 30 min prior to the time point and processed for EdU detection and telomere-FISH (telomere-PNA probe). Foci containing both EdU and telomere signals were scored as telomere replication foci. Percentages of telomere replication signals over total telomere signals were obtained. Data were obtained from three independent experiments, and error bars represent standard deviations. At least 400 foci were counted for each experiment. (B) Representative image of stained cells with replicating (colocalized EdU and telomere signals) and non-replicating (not colocalized) telomere foci are shown. (C) Timeless depletion caused a mild delay in bulk BrdU incorporation. To assess the rate of DNA replication, cells used in A were also incubated with BrdU for 30 min at the indicated times after the release from a double thymidine block. Genomic DNA was fixed on nitrocellulose membrane to monitor the incorporation of BrdU by western blotting using an anti-BrdU antibody. Relative intensities of signals are shown below. Asy: Asynchronous cells. (D) Cell cycle analysis of cells used for telomere replication assays in (A and B).