Dock11 knockout ameliorates cellular and functional defects in HSCs caused by DCAF8 deficiency. (A) Immunoblot of GTP-bound (active) CDC42 and total CDC42 in Lin⁻ cells from WT, Dcaf8^−/−, Dcaf8^−/−Dock11^−/−, and Dock11^−/− mice, and (B) the ratio of GTP-bound CDC42 to total CDC42 (n = 3 independent experiments). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (C) Representative immunofluorescence images showing the distribution of CDC42 and tubulin in purified LT-HSCs from WT, Dcaf8^−/−, and Dcaf8^−/−Dock11^−/− mice. Scale bar, 5 μm. (D) Percentages of LT-HSCs with polarized distribution of CDC42 and tubulin in WT, Dcaf8^−/−, and Dcaf8^−/−Dock11^−/− mice (n = 3 independent experiments, in each experiment at least 40 cells pooled from 3 mice were analyzed). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (E-H) Representative images of (E) P16 and (G) P21 indicated by immunofluorescence, with quantification of (F) P16 (n = 20 cells, pooled from 3 mice) and (H) P21 (n = 40, pooled from 3 mice) by MFI in purified LT-HSCs from WT, Dcaf8^−/−, and Dcaf8^−/−Dock11^−/− mice. Scale bar, 5 μm. Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (I) Representative images of γ-H2AX indicated by immunofluorescence, with (J) quantification of γ-H2AX foci in purified LT-HSCs from WT (n = 53 cells, pooled from 3 mice), Dcaf8^−/− (n = 67 cells, pooled from 3 mice), and Dcaf8^−/−Dock11^−/− mice (n = 63 cells, pooled from 3 mice). Scale bar, 5 μm. Statistical analysis used Kruskal-Wallis test followed by the Dunn multiple comparisons test. (K) Representative images of SA-β-gal staining and (L) quantification in the BM cells from WT, Dcaf8^−/−, and Dcaf8^−/−Dock11^−/− mice (n = 3 independent experiments, each pooled from 3 mice). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (M) Total number of colonies after 1 × 10⁴ BM cells were plated to determine the colony-forming capacity of WT, Dcaf8^−/−, and Dcaf8^−/−Dock11^−/− mice (n = 4 mice). Statistical analysis used 1-way ANOVA followed by the Fisher least significant difference test. (N) Schematic of the transplantation assay as illustrated in panels O-Q. (O) Donor chimerism analyses in the PB of recipients at the indicated time points (n = 9 recipients for WT and Dcaf8^−/−Dock11^−/−, n = 8 recipients for Dcaf8^−/−). Statistical analysis used 2-way ANOVA followed by the Fisher least significant difference test. (P) Donor chimerism analyses in the PB of recipients at the end point (n = 9 recipients for WT and Dcaf8^−/−Dock11^−/−, n = 8 recipients for Dcaf8^−/−). Statistical analysis used 2-way ANOVA followed by the Fisher least significant difference test. (Q) 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 = 9 recipients for WT and Dcaf8^−/−Dock11^−/−, n = 8 recipients for Dcaf8^−/−). Statistical analysis used 1-way ANOVA followed by the Fisher least significant difference test. For panels J and O, data are presented as 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; ns, no statistical significance; T, T cells.