Knockout of p16 and/or Arf has no effect on TBI-induced HSC senescence and long-term injury. (A-C) Two months after 6 Gy TBI, total BM cells (BMCs) were harvested from control and irradiated (TBI) p16^−/−, Arf^−/−, and p16Arf^−/− mice and p16^+/+, Arf^+/+, and p16Arf^+/+ mice for clonogenic function analysis by CAFC assays. The numbers of 1-, 2-, 4-, and 6-week CAFCs were counted and expressed as mean ± SD (n = 3 mice/group) of CAFCs per 100 000 BMCs. *P < .05 and **P < .01, TBI vs CTL. (D) Two months after 6 Gy TBI, total BM cells (BMCs) were harvested from control and irradiated (TBI) p16Arf^−/− and p16Arf^+/+ mice for analyses of SA-β-gal activity in various populations of BM hematopoietic cells using C₁₂FDG as a substrate. Percentages of SA-β-gal–positive cells in HPCs, LSK cells, MPPs, HSCs, ST-HSCs, and LT-HSCs are presented as mean ± SD (CTL: n = 3 and TBI: n = 4). **P < .01 and ***P < .001, TBI vs CTL. (E) Two months after 6 Gy TBI, total BM cells (BMCs) were harvested from control and irradiated (TBI) p16^−/− and p16^+/+ mice for analysis of HSC long-term repopulating ability by CRA as described in Figure 2B. Percentages of total donor-derived (CD45.2) hematopoietic cells in peripheral blood of the recipients and percentages of T cells, B cells, and myeloid (M) cells in donor-derived hematopoietic cells in the peripheral blood of recipients are presented as mean ± SD (n = 9-10 recipients from 2 independent experiments). ***P < .001, TBI vs CTL. (F) Repopulating units (RU) calculated according to the engraftment data presented in (E) are presented as mean ± SD. ***P < .001, TBI vs CTL.