Figure S1.

Extended characterization of HSC functionality upon Ebf1 deficiency. Related to Figs. 1 and 2. (A) Gating strategies for HSC, MPP2, MPP3, and MPP4 cells. (B) Gating strategies for CLP, ALP, and BLP cells. Representative plots are shown for Ebf1^WT and Ebf1^KO mice. (C) Frequencies of myeloid cells (CD11b⁺), B cells (CD19⁺), and T cells (CD4⁺ CD8⁺) within CD45.1 donor cells in the BM of the Ebf1^WT and Ebf1^KO recipients 16 wk after AdT. Ebf1^WT n = 13, Ebf1^KO n = 14. (D) Absolute number of CD41⁺ HSCs in the BM. Ebf1^WT n = 11, Ebf1^KO n = 9, Ighm^WT n = 11, and Ighm^Tm1 n = 14. (E) 300 WT CTY-labeled HSCs were cultured in TPO and SCF, with increased concentrations of BM fluid collected from Ebf1^WT and Ebf1^KO mice. Division index (CTY mean fluorescence intensity [MFI] from day n/CTY MFI from day 0) at day 2 and day 5, of CTY-labeled HSCs cultured with different BM fluid concentrations from Ebf1^WT and Ebf1^KO mice, n = 6–8. (F) CFU assay performed with 150 sorted HSCs plated in triplicate in methocult. Total number of colonies formed after 10–12 d of plating. Biological replicates Ebf1^WT and Ebf1^KO n = 2, Ighm^WT and Ighm^Tm1 n = 3. (G) CFU assay performed with 150 sorted LT-HSCs (LSK CD34⁻Flt3⁻CD150⁺CD48⁻) plated in triplicate in methocult. Colony identification after 10–12 d of plating. Biological replicates Ebf1^WT and Ebf1^KO n = 5. (H) Frequency of CD45.2 donor-derived cells in the peripheral blood within T cells (CD4⁺ CD8⁺; ****, P < 0.0001; ***, P = 0.0004; **, P = 0.0013), myeloid cells (CD11b⁺; ***, P = 0.0002; ****, P < 0.0001; **, P = 0.0010; **, P = 0.0024) and B cells (CD19⁺; **, P = 0.0060; ****, P < 0.0001), during the AdT. Data are represented as mean ± SEM. Ebf1^WT n = 33 and Ebf1^KO n = 40, Ighm^WT n = 17, and Ighm^Tm1 n = 19. (C–H) Statistical significance was determined by Mann-Whitney U test. Data are from >2 independent experiments.