Figure 3
Figure 3. Cell-cycle arrest and increase of aging-related genes in TERT-deficient HSCs. (A) Foci of chromatin condensation in TERT−/− and ATM−/−TERT−/− murine HSCs. CD34−LSK cells from the indicated genotypes were stained with anti-H3K9Me Ab (top) or anti-HP1γ Ab (bottom). Nuclei were detected using DAPI staining (magnification ×1800). (B) qPCR analysis of proliferation-related genes in CD34− LSKs from mice of the indicated genotypes. Reduced expression of cyclin A2 in aged TERT−/− murine HSCs and PCNA in aged TERT−/− and ATM−/−TERT−/− murine HSCs (bottom), which is not seen in young HSCs (top). Data shown are the mean ratio (± SD) of mRNA to β-actin levels (*P < .05, n = 4). (C) Cell-cycle status of freshly isolated LSKs from mice of the indicated genotypes, assayed by Ki67 staining. TERT−/− and ATM−/−TERT−/− LSKs showed increased Ki67-negative populations, whereas ATM−/− LSKs showed a reduced Ki67-negative population. Data shown are representative FACS patterns derived from 3 independent experiments (left); graphs show the mean (± SD) percentage of Ki67-negative cells (right; *P < .05). (D) Cell-cycle status of LSKs of each murine genotype, as evaluated by BrdU short-labeling assay. BrdU was administered for 24 hours to mark cells that entered S phase, and Hoechst was administered to detect DNA content. Representative FACS patterns derived from 3 independent experiments (left) and graphs showing the mean (± SD) percentage of BrdU-negative or BrdU-low/positive cells (right) are shown (*P < .05). TERT−/− and ATM−/−TERT−/− LSKs showed relatively increased BrdU-negative populations and reduced BrdU-low/positive populations, whereas ATM−/− LSKs showed reduced BrdU-negative populations and increased BrdU-low/positive populations. (E) qPCR analysis of p16INK4a, p19ARF, and p21 in LSKs from aged mice of the indicated genotypes (37 weeks of age). Increased expression of p16INK4a and p19ARF in TERT−/− and ATM−/−TERT−/− murine HSCs and reduced expression of p21 in these HSCs. Data shown are the mean ratio (± SD) of mRNA to β-actin levels (*P < .05, n = 3).

Cell-cycle arrest and increase of aging-related genes in TERT-deficient HSCs. (A) Foci of chromatin condensation in TERT−/− and ATM−/−TERT−/− murine HSCs. CD34−LSK cells from the indicated genotypes were stained with anti-H3K9Me Ab (top) or anti-HP1γ Ab (bottom). Nuclei were detected using DAPI staining (magnification ×1800). (B) qPCR analysis of proliferation-related genes in CD34− LSKs from mice of the indicated genotypes. Reduced expression of cyclin A2 in aged TERT−/− murine HSCs and PCNA in aged TERT−/− and ATM−/−TERT−/− murine HSCs (bottom), which is not seen in young HSCs (top). Data shown are the mean ratio (± SD) of mRNA to β-actin levels (*P < .05, n = 4). (C) Cell-cycle status of freshly isolated LSKs from mice of the indicated genotypes, assayed by Ki67 staining. TERT−/− and ATM−/−TERT−/− LSKs showed increased Ki67-negative populations, whereas ATM−/− LSKs showed a reduced Ki67-negative population. Data shown are representative FACS patterns derived from 3 independent experiments (left); graphs show the mean (± SD) percentage of Ki67-negative cells (right; *P < .05). (D) Cell-cycle status of LSKs of each murine genotype, as evaluated by BrdU short-labeling assay. BrdU was administered for 24 hours to mark cells that entered S phase, and Hoechst was administered to detect DNA content. Representative FACS patterns derived from 3 independent experiments (left) and graphs showing the mean (± SD) percentage of BrdU-negative or BrdU-low/positive cells (right) are shown (*P < .05). TERT−/− and ATM−/−TERT−/− LSKs showed relatively increased BrdU-negative populations and reduced BrdU-low/positive populations, whereas ATM−/− LSKs showed reduced BrdU-negative populations and increased BrdU-low/positive populations. (E) qPCR analysis of p16INK4a, p19ARF, and p21 in LSKs from aged mice of the indicated genotypes (37 weeks of age). Increased expression of p16INK4a and p19ARF in TERT−/− and ATM−/−TERT−/− murine HSCs and reduced expression of p21 in these HSCs. Data shown are the mean ratio (± SD) of mRNA to β-actin levels (*P < .05, n = 3).

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