FIG 10.
RCT-L247E rescues telomere deprotection phenotypes of TIN2 deletion. (A) Growth curve of SV40-LT-immortalized TIN2−/− MEFs expressing an empty vector (EV) or the indicated FLAG-HA2 RCT-TIN2 alleles. (B) Examples of metaphases in TIN2-deficient cells expressing RCT-TIN2 alleles by telomeric FISH. Green, TelC PNA probe; red, DAPI DNA stain. (C) Summary of telomere phenotype TIN2-deficient cells expressing RCT-TIN2 alleles, as determined by FISH, as shown in panel B. Values are averages and SDs from 3 independent experiments. Chromosome-type and chromatid-type telomere fusions were scored on 3,000 to 5,000 telomeres/experiment. Long-arm sister telomere fusions and fragile telomeres were scored on 2,000 to 3,000 telomeres/experiment. (D) IF for 53BP1 (red) and telomeric FISH (green) in TIN2−/− MEFs (72 h post-Cre) transduced with RCT-TIN2 or RCT-L247E. DNA is stained with DAPI (blue). Bars, 1.5 μm. (E) Quantification of 53BP1 TIFs, as determined for panel D (n > 100 nuclei) after Cre (72 h). Averages and median values are from three independent experiments and SDs. **, P < 0.05 (paired Student's t test). (F) Immunoblots for Chk1 and Chk2 phosphorylation in TIN2F/F MEFs expressing the indicated FLAG-HA2 TIN2 alleles and treated with Cre (72 h) as indicated.