Twist1 cKO animals and HSCs are sensitized to IR and 5-FU. (A) Kaplan-Meier survival curves of mice after IR at 7 Gy (n = 6; log-rank test). (B) Frequencies of apoptotic cells in the LT-HSCs from mice at basal and 12 days after IR (n = 4; Student t test). (C) Colony-forming capacity of LT-HSCs. LT-HSCs from cKO and control (Ctrl) mice, untreated (no IR) or after IR (2 Gy), were plated in methylcellulose-containing media, and colonies were counted and classified as GEMM, GM, G, M, or BFU-E on day 10 of culture (n = 4; Student t test). (D) Quantification of γ-H2AX foci (green) in nuclei (blue) of CD34⁻ LSK cells without IR and at the indicated times post-IR at 2 Gy (right) and representative confocal microscopic images at 1 hr after IR (left). Scale bars, 10 µm (Student t test). (E) Percentages of SA-β-gal staining in LT-HSCs at basal and 12 days after IR at 5 Gy (right) and representative fluorescence-activated cell sorting (FACS) plots (left; n = 4-5; Student t test). (F) The MFI of p16 expression in LT-HSCs under steady-state conditions and after IR and representative FACS plots (left; n = 5-6; Student t test). (G) Absolute leukocyte count in PB after a single injection of 5-FU (n = 4-6; Student t test). (H) Kaplan-Meier survival curves of mice after serial doses of 5-FU treatment at 10-day intervals (n = 11-12; log-rank test). (I) Cell-cycle analysis of LT-HSCs in BM after 5-FU treatment (n = 4; Student t test). (J) Percentages of SA-β-gal staining in LT-HSCs at day 16 after 5-FU injection (n = 5). (K) The MFI of p16 expression in LT-HSCs after 5-FU treatment (n = 5). Data are shown as means ± standard deviation. *P < .05, **P < .01, ***P < .001. DAPI, 4′,6-diamidino-2-phenylindole; MFI, mean fluorescence intensity; ns, not significant; WBC, white blood cell.