TRF1 deletion leads to increased number of TIFs and cellular senescence via p21 but no induction of apoptosis. (A) Representative image of colocalization (indicated by white arrows) of γH2AX foci and telomeres using immuno Q-FISH. (B) Quantification of the number of TIFs per detected nucleus. Only cells showing 3 or more TIF were included. Mice without pI-pC–induced Cre expression are represented by dark gray bars and mice undergoing pI-C injections by light gray bars. Two-sided t test was used for statistical comparison. (C) Western blot analysis of p53 protein levels of pI-pC–treated TRF1^flox/floxMx1-wt and TRF1^flox/floxMx1-Cre mice. (D) Quantification of Western blot analysis of the p53 protein levels in relation to respective actin levels. Two-sided t test was used for statistical comparison. (E) Fold change of quantitative PCR of p21 mRNA levels in FACS-sorted bone marrow cells sorted for HSC: lin(−), c-kit (+), sca-1(+); progenitor cells: lin(−), c-kit(+), sca-1 (−); and differentiated cells: lin(+). mRNA of p21 levels were normalized to actin levels, and statistical comparison was conducted with the Student t test between untreated and pI-pC–treated TRF1^flox/floxMx1-wt and TRF1^flox/floxMx1-Cre mice. (F) Representative image of p21 IHC staining of TRF1^flox/floxMx1-Cre mice with and without pI-pC injections (×40 magnification, blue bar represents 50 μm). (G) Quantification of the p21 IHC-positive area (brown cells) in relation to the area of all cells. Mice without pI-pC–induced Cre expression are represented by dark gray bars and mice undergoing pI-C injections by light gray bars. Two-sided t test and Student paired t test was used for statistical comparison between TRF1^flox/floxMx1-wt and TRF1^flox/floxMx1-Cre subgroups. (H) Representative images of β-galactosidase staining in unsorted bone marrow. Images were captured with ×10 magnification, small window represents a magnified (20×) section of the image showing representative β-galactosidase–positive (indicated by black arrows) and –negative cells. (I) Quantification of the percentage of β-galactosidase positive cells per counted dish. Bar graph on the left represents unsorted bone marrow cells. On the right, quantification of FACS-sorted lin(+) differentiated cells and lin(−)c-kit(+) HSC and progenitors cells is shown. Mice without pI-pC–induced Cre expression are represented by dark gray bars and mice undergoing pI-C injections by light gray bars. Two-sided t test was used for statistical comparison. (J) Quantification of the percentage of early apoptotic cells in all bone marrow cells and stem and progenitor cells. Quantification on the left represents all bone marrow cells, on the right, quantification of lin(−)c-kit(+) HSC and progenitors cells is shown. Mice without pI-pC–induced Cre expression are represented by dark gray bars, mice undergoing pI-C injections by bright gray bars. Two-sided t test and Student t test was used for statistical comparison between TRF1^flox/floxMx1-wt and TRF1^flox/floxMx1-Cre subgroups. (K) Representative FACS analysis for apoptosis of pI-pC–treated TRF1^flox/floxMx1-wt and TRF1^flox/floxMx1-Cre mice. Single cells in Ki were further gated on the basis of forward and side scatter (Kii). Selected cells of Kii were analyzed as total bone marrow based on TO-PRO-3 and annexin-V staining or further separated in panel Kiii on the basis of negative linage staining. Lineage(−) cells were further distinguished in c-kit–positive and –negative cells (in Kiv). C-kit(+), lin(−) HSC, and progenitor cells (Kiv) were further analyzed on the basis of TO-PRO-3 and annexin-V staining. Annexin-V–positive and TOP-RO-3–negative cells were identified as early apoptotic cells.