FIG. 4.

Cell cycle status and telomere integrity in ER-POT1 cells. (A and B) Size and mitotic index of ER-POT1 cells. (A) Phase-contrast images of ER-POT1 cells grown with or without tamoxifen (+Tam or −Tam, respectively) for 24 h. (B) Staining of metaphase cells with phospho-H3 antibody and DAPI. Cells were grown with or without tamoxifen for 20 h (left panels); caffeine (Caf) and nocodazole (Noc) were then added and cells harvested 1 h later (right panels). (C) Telomeric dysfunction-induced foci induced by POT1 removal. Cells were grown with or without tamoxifen for 16 h, fixed, and stained with γH2AX primary and RRX-conjugated secondary antibody. Cells were refixed, and telomeric DNA was visualized by FISH using fluorescein isothiocyanate-conjugated PNA probe. Note that only a fraction of the FISH signals correspond to actual telomeres, because chicken cells contain large blocks of interstitial telomeric DNA (53). (D) Localization of TRF1 and RAP1 after POT1 removal. Cells were grown with or without tamoxifen for 48 h, fixed, and stained with DAPI and antibody to TRF1 or RAP1.