Functional failure of Zbtb11^hKO HSC: failure to home after transplantation, failure to proliferate in vitro, and impairment of cell cycle progression. (A) Schematic description of the transplantation and homing analysis. (B) Homing efficiency of E16.5 Zbtb11^hKO and WT FL HSCs transplanted into WT neonate. (C) Schematic description of the in vitro proliferation assay in which 75 immunophenotyped Zbtb11^hKO or WT FL HSCs were seeded per well and cultured in growth medium. Cells were counted in each well on day 7 and divided by 75 to determine the fold expansion. (D) Flow cytometry quantification of in vitro proliferation assays: on day 7, E14.5 HSC culture resulted in mean = 133 Zbtb11^hKO cells and 85 078 control cells, respectively; the average fold expansion was 1.78 and 1142.7 over initial seeding of 75 cells. (E) Schematic description of the cell cycle analysis. Cell cycle progression was assessed using the Ki67/nuclear DNA (Hoechst) ratio for (F) LSK and (G) granulocytes (Gr1⁺) at E17.5. For panel B, n = 2 independent experiments. For panel D: E14.5 WT, n = 6 and KO, n = 6; E17.5 WT, n = 2 and KO, n = 2. For panels F and G: WT, n = 5 and KO, n = 3; WT/controls (green); KO, Zbtb11^hKO (red); data ± standard error of the mean. Two-way analysis of variance with Šídák multiple comparisons: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.