Alteration of telomeres is unlikely to be responsible for the impairment of the HSC pool seen in ATM and TERT doubly deficient mice. (A) Increased γH2AX foci in aged ATM^−/− and TERT^−/− LSKs, and significantly further increased γH2AX foci in aged ATM^−/− TERT^−/− LSKs (bottom). In young ATM^−/− and ATM^−/−TERT^−/− mice, γH2AX foci are only mildly increased (top). LSKs from mice of the indicated genotypes were stained with anti-γH2AX Ab (red), and TOTO3 staining was used to detect nuclei (blue; left: magnification ×600). The numbers of γH2AX foci in each cell were counted (right; *P < .05, **P < .01). (B) Relative telomere length of LSKs (left), measured by flow FISH. There are no apparent differences in telomere length among the different genotypes. Original FACS plots of flow FISH are shown (right). The 2 peaks in each plot are correspondent to signals in cells labeled with the telomere PNA probe and in unlabeled cells as a control. (C) Telomere FISH analysis of cells in metaphase. Representative pictures (top: magnification ×5000) and frequencies of telomere signals per cell (bottom) are shown. No alteration of telomere signals was detected in cells from mice of any of the genotypes. (D) No significant increase of TIFs in LSKs of all genotypes. LSKs from the indicated genotypes were costained with anti-TRF1 Ab (green) and anti-53BP1 Ab (red), nuclei were detected using DAPI staining (blue: left: magnification ×7000). The mean frequency (± SD) of merged yellow signals in each cell was determined by counting (right; *P < .05).