Figure 1
Heterozygous loss of Trf1 and Tin2 leads to a dose-dependent reduction in expression, affects their binding to telomeres and leads to the enhanced formation of tumors during aging. (a and b) Quantitative analysis of Trf1 (a) and Tin2 (b) mRNA levels normalized to hydroxymethylbilane synthase in total spleen extracts of 14–16-month-old mice of the indicated genotypes. (c) Quantification of Tin2 protein expression levels relative to GAPDH by western blot analysis in total spleen protein extracts of 14–16-month-old mice of the indicated genotypes. (d) Trf1+/− and Tin2+/− mice were crossed with HA-tagged Trf1 knockin mice (Trf1ki/ki). Western blot detection of a N-terminal HA-tagged version of Trf1 (HAmTrf1) and mTin2 after immunoprecipitation of HATrf1 with an HA-specific antibody from protein extracts of testis of mice of the indicated genotypes. Western blot analysis of p84 was used as loading control. (e) Immuno-fluorescence in situ hybridization (FISH) staining of telomere-binding proteins (Trf1 and Tin2) and telomeric ends in mouse embryonic fibroblasts (MEFs) of the indicated genotypes. FISH of telomeric repeats was conducted using a [TTAGGG]3-Cy3 peptide nucleic acid (PNA) probe (red). Specific antibodies against the Shelterin proteins were used in combination with a secondary Alexa-488 labeled antibody (green). (f and g) Percentage of co-localization of Trf1 (f) and Tin2 (g) foci at telomeric ends from MEFs of the indicated genotypes. (h) Kaplan–Meyer curves showing survival of wild-type mice (n=26), Tin2+/− mice (n=21), Trf1+/− mice (n=32) and Trf1+/−Tin2+/− mice (n=33). The overall survival rate of the indicated genotypes is not significantly affected during the observation period of 140 weeks. (i) Quantification of the hematopoietic stem and progenitor cell compartment in 14–16-month-old mice by fluorescence activated cell sorting of the indicated genotypes. The percentages were calculated for viable in total bone marrow (panel I–III) (n=4–8 mice per group). (j) Kaplan-Meyer curves showing tumor-free survival of the indicated mouse cohorts. Note that the heterozygous deletion of Trf1 and/or Tin2 reduced the latency of tumor formation. 18–24-month-old Trf1+/− mice, Tin2+/− mice and Trf1+/−Tin2+/− mice showed increased rates of tumor formation compared with wild-type mice developing tumors at 24–30 month. (k) Spectrum of the histological analyzed tumors. Malignant tumor formation is significantly enhanced in the knockout mice compared with the wild-type mice. Error bars indicate s.d. and the Student t-test was used for statistical calculations. (h and j) the Mantel–Cox test was used for calculations of P-values.
