Figure 2
Trf1 and Tin2 deficiency leads to increased telomere fragility and sister chromatid exchange accompanied by an accumulation of DNA damage at telomeric ends in vivo. Metaphase spreads from freshly isolated bone marrow cells were analyzed by fluorescence in situ hybridization (FISH) using telomere specific probes: (a) the telomere fluorescence intensity (TFI) was quantified from 410 cells of wild-type mice and 728 cells of Trf1+/−Tin2+/− mice. The dot plot shows the mean TFI of individual nuclei. (b) Representative images of telomere fluorescence (Q-FISH) distribution in metaphase spreads from Trf1+/− Tin2+/− mice using a [TTAGGG]3-Cy3 peptide nucleic acid (PNA) probe. High magnifications on the right show telomere fragility characterized by multitelomeric signals (lower picture) or broken telomeres (upper picture). (c and d) Quantification of the percentage of broken telomeres (c) and multitelomeric signals (d) per metaphase from bone marrow cells of the indicated genotypes. 144 individual metaphases of wild-type mice, 294 metaphases of Trf1+/−Tin2+/− mice, 204 metaphases of Trf1+/− mice and 236 metaphases of Tin2+/− mice have been analyzed. (e) Metaphase spreads from Trf1+/− Tin2+/− mouse embryonic fibroblasts (MEFs) were analyzed by telomeric CO-FISH using a [TTAGGG]3-Alexa488 PNA probe (green) and a [CCCTAA]3-Cy3 PNA probe (red). High magnifications on the right show telomere fragility characterized by chromosomes with sister chromatid fusions (upper left), multitelomeric signals (upper right) or sister chromatid exchange (lower left). (f) Frequency of sister chromatid exchanges in metaphase spreads of MEFs from the indicated genotypes. 297 individual metaphases of wild-type mice, 316 metaphases of Trf1+/−Tin2+/− mice, 301 metaphases of Trf1+/− mice and 307 metaphases of Tin2+/− mice have been analyzed. (g and h) Analysis of 53BP1 foci in the basal crypts of the small intestine from 14–16-month-old mice of the indicated genotypes: (g) representative images of 53BP1 foci in the basal crypts, (h) percentage of cells showing 53BP1 signals in the nuclei of crypt cells. In total, cells from 185 crypts of wild-type mice, 229 crypts of Trf1+/−Tin2+/− mice, 149 crypts of Trf1+/− mice and 146 crypts of Tin2+/− mice have been analyzed. (i and j) Analysis of γ-H2AX foci in the basal crypts of the small intestine from 14–16-month-old mice of the indicated genotypes: (i) representative images of γ-H2AX foci in the basal crypts, (j) percentage of cells showing γ-H2AX signals in the nuclei of crypt cells. In total, cells from 244 crypts of wild-type mice, 230 crypts of Trf1+/−Tin2+/− mice, 231 crypts of Trf1+/− mice and 245 crypts of Tin2+/− mice have been analyzed. (k and l) Immuno-FISH analysis of γ-H2AX foci at telomeres using a γH2AX antibody (green) and a [TTAGGG]3-Cy3 PNA probe (red): (k) representative images of γH2AXat telomeres in crypts of Trf1+/− Tin2+/− knockout mice, (l) percentage of cells with co-localization of γ-H2AX foci at telomeres. n=number of independent MEF cultures used per genotype. Error bars indicate s.d. and P-values are indicated. (c, d, f and j) the Mann–Withney test was used for calculations of P-values. (h and l) The Student t-test was used for calculations of P-values.
