Figure 5.

CTC1 deletion activates a DDR. (A) Immunoblot of pATR, p53 and p21 in WT MEFs treated with the indicated shRNAs. γ-Tubulin served as loading control. (B) Immunoblot of pATM, pChk1 and pChk2 in two independent lines of MEFs of the indicated genotypes. γ-Tubulin served as loading control. (C) Immunoblot of pATM, pChk1, pChk2, Flag–CTC1, HA–STN1 and endogenous STN1 in CTC1 null MEFs reconstituted with indicated cDNAs. γ-tubulin served as loading control. (D) Proliferation of SV40 immortalized CTC1 null MEFs reconstituted with the indicated cDNAs. (E) Telomere-PNA FISH demonstrating the localization of shelterin components to telomeres in CTC1^−/− MEFs. Cells were stained with the indicated antibodies (green), telomere-PNA FISH (red) and DAPI (blue). Localization of the indicated shelterin proteins on telomeres (telo) in CTC1 null MEFs. (F) γ-H2AX-positive TIFs in WT (top) and CTC1^−/− (bottom) MEFs, expressing either vector or TPP1^ΔRD cDNA. Cells were fixed 72 h after retroviral infection, stained with anti-γ-H2AX antibody (green), Tam-OO-(CCCTAA)₄ telomere peptide nucleic acid (red) and DAPI (blue). (G) WT MEFs expressing TPP1^ΔRD or CTC1^−/− MEFs were arrested in mitosis with colcemid, metaphase spreads prepared and stained with anti-γ-H2AX antibodies (green), Tam-OO-(CCCTAA)₄ telomere peptide nucleic acid (red) and DAPI (blue). Arrows point to sites of γ-H2AX localization. Insets show enlarged images. (H) CTC1^−/− MEFs (two independent lines), WT MEFs treated with shRNA against TRF2 or expressing TPP1^ΔRD were assayed for TIF formation. Quantification of the number of TIFs with or without visible telomere signals co-localizing with γ-H2AX staining are indicated.