Dcaf8 knockout leads to impaired self-renewal capacity of HSCs. (A) Total number of colonies after 1 × 10⁴ BM cells were plated for 2 rounds to determine the colony-forming capacity (n = 3 mice). (B) Number of colonies divided by type after the second-round plating as shown in panel A (n = 3 mice). (C) Schematic of the transplantation assay as illustrated in panels D-E. (D-E) Donor chimerism analyses in the PB of (D) primary recipients (n = 6 recipients) and (E) secondary recipients (n = 7 recipients for WT group, n = 10 recipients for Dcaf8^−/− group) at the indicated time points. Statistical analysis used 2-way ANOVA followed by the Fisher least significant difference test. (F) Schematic of the competitive transplantation assay as illustrated in panels G-H. (G) Ratio of donor chimerism to competitive mice in the PB of the recipients at the indicated time points (n = 6 recipients for WT group; n = 8 recipients for Dcaf8^−/− group). Statistical analysis used 2-way ANOVA followed by the Fisher least significant difference test. (H) Ratio of donor chimerism to competitive mice in the BM of the recipients at the end point (n = 4 recipients). (I) Schematic of the LT-HSC competitive transplantation assay as illustrated in panels J-K. (J) Donor chimerism analyses in the PB of the recipients at the indicated time points (n = 8 recipients for WT group; n = 9 recipients for Dcaf8^−/− group). Statistical analysis used 2-way ANOVA followed by the Fisher least significant difference test. (K) Frequencies of myeloid (CD11b⁺), neutrophils (CD11b⁺Ly6G⁺), B cells (CD19⁺), and T cells (CD3⁺) in donor-derived cells in the PB of the recipients at the end point (n = 8 recipients for WT group; n = 9 recipients for Dcaf8^−/− group). (L) The Kaplan-Meier survival curve of WT and Dcaf8^−/− mice after 2 injections of 5-FU (150 mg/kg) administered 7 days apart (n = 8 mice). P value was calculated by log-rank test. (M) The Kaplan-Meier survival curve of WT (n = 47 mice) and Dcaf8^−/− (n = 34 mice) mice. P value was calculated by log-rank test. For panels D, E, G, and J, data are presented as mean ± standard error of the mean (SEM); other data are presented as mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. B, B cells; BFU-E, burst-forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GM, colony-forming unit-granulocyte, macrophage; ns, no statistical significance; T, T cells; Tx, transplantation.