ATF4 depletion in hematopoietic cells impairs HSC self-renewal. (A) Experimental workflow showing that Mx1-Cre;Atf4^fl/fl (Δ/Δ) and Atf4^fl/fl (fl/fl) mice were given pIpC and euthanized for analysis 1 month later. (B) qRT-PCR analysis of Atf4 expression in BM cells of fl/fl and Δ/Δ mice (n = 4 mice). (C) BM cell numbers in fl/fl and Δ/Δ mice (n = 4-5 mice). (D-H) Percentages of indicated cell populations in the BM of fl/fl and Δ/Δ mice (n = 4-5 mice). (I) Experimental workflow for serial and competitive transplantation. (J) Percentage of CD45.2⁺ donor cells in the PB of recipient mice at the indicated time points after competitive BM transplantation (n = 5-8 mice). (K) Percentage of CD45.2⁺ donor-derived cells in the BM of recipients (n = 3-4 mice). (L-M) Percentages of different CD45.2⁺ donor–derived cell populations in the BM of recipients (n = 3-4 mice). (N) Percentages of CD45.2⁺ donor cells in the PB of recipient mice at the indicated time points after secondary BM transplantation (n = 7 mice). (O) Results from CFU colony assays performed using 150 LKS⁺ cells from fl/fl and Δ/Δ mice cultured for 10 to 14 days in complete methylcellulose-based medium (n = 4-5 wells for 3 independent experiments). Data represent mean ± standard deviation (SD). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; unpaired 2-tailed Student t test.