Gadd45a^−/−HSCs exhibit impaired apoptosis and delayed DNA repair after IR. (A) Single HSCs (CD34⁻LSK) were sorted in 96-well plates, followed by 1- to 2-Gy IR and culture for 14 days. Colonies were counted in each group (n = 4). (B) The cell cycle profile of HSCs (Flt3⁻LSK) was analyzed using BrdU incorporation assay 24 hours after 2-Gy IR (n = 3). (C) IR-induced apoptosis was detected with Annexin V/DAPI staining 24 hours after 1- to 2-Gy IR. The early apoptosis (Annexin V⁺/DAPI⁻) and late apoptotic cells (Annexin V⁺/DAPI⁺) are shown (n = 4). (D) γ-H2AX foci per cell were counted at the indicated time point post-IR (HSC, n = 40; HPC, n = 60). Representative immunofluorescence staining for γ-H2AX and the nucleus is shown. (E) Survival curves showed the survival rate of mice subjected to 4.5-Gy IR twice (at a 3-month interval) for the indicated genotypes (n = 9). (F-G) The cell cycle status and apoptosis were analyzed 4.5 months after the second 4.5-Gy IR (n = 3) (*P < .05, **P < .01). N.S., not significant.