FIGURE 4.

Loss of ATF3 impairs the long-term self-renewal of HSCs. (A) A total of 1 × 10³ LT-HSCs from Atf3^fl/fl and Atf3^fl/flVav1Cre mice (CD45.2⁺) was mixed with competitor BM cells (CD45.1⁺) and injected into lethally irradiated recipients (CD45.1/2⁺). Four months later, the chimeric BM cells were re-transplanted into secondary or tertiary recipients (CD45.1/2⁺) (1°/2°/3° defined as primary, secondary, and tertiary transplantations, n = 6). Percentages of donor-derived cells in the peripheral blood (PB) were analyzed by flow cytometry at the indicated time points during serial BMT (n = 6). (B) Analysis of distinct lineages (CD3⁺ T cells, B220⁺ B cells, and Gr-1⁺ myeloid cells) in PB from recipients 4 months after transplantation (n = 6). (C) Absolute numbers of donor-derived LT-HSCs in the BM from recipients 4 months after transplantation (n = 6). (D) Percentages of donor-derived LT-HSCs in the BM cells in different cell cycle stages from recipients at 4 months after 3° transplantation (n = 6). (E) The percentages of BrdU⁺ LT-HSCs in the BM from recipients at 4 months after 3° transplantation (n = 6). (F) Survival curve of Atf3^fl/fl and Atf3^fl/flVav1Cre recipient mice after transplantation (n = 28). (G) Homing analysis of CFSE⁺ LSKs from Atf3^fl/fl and Atf3^fl/flVav1Cre BM cells 16 h after transplantation (n = 6). (H) At 4 months after 2°BMT, 1000 sorted LSKs from recipients were seeded in a colony-forming unit assay (n = 6). Data are representative of two independent experiments. Error bars show the mean ± SD. P values were determined using two-sided Student’s t tests. *P < 0.05; **P < 0.01.