Disruption of the β-catenin-TCF/LEF transcription-mediating complex expands HSPCs and diminishes neutrophilic production. (A) Quantitative RT-PCR of whole BM from WT (wt), heterozygous (het), and homozygous (dn) dnTCF4 mice. The y-axis represents relative dnTCF4 expression compared with Gapdh control. Graph shows representative experiment out of 2 independent experiments (n = 5). (B) Western blot analysis for TCF4 and dnTCF4 expression in WT (wt) and dnTCF4 (dn) mouse BM. Membrane was stained with antihuman TCF4 antibody to detect TCF4 and dnTCF4 protein. β-actin expression was used as a loading control. Positions of molecular weight standards are indicated (kDa). (C) Quantitative RT-PCR in BM c-Kit⁺ cells isolated from WT^T (wt^T) and dnTCF4 (dn) mice. The y-axes represent Axin2 and Nkd1 expression relative to Gapdh upon treatment with 10 μM CHIR99021 (a GSK3β inhibitor that prevents β-catenin phosphorylation and favors its translocation to the nucleus, inducing transactivation of β-catenin target genes). The x-axis indicates hours after stimulation (h); n = 3 mice per time point per group. (D) Representative flow cytometry plots from 1 WT (upper plots) and 1 dnTCF4 (lower plots) mouse. Plots illustrate gating strategy. Color boxes indicate the following populations: Lin⁻ cells (gray), Lin⁻ c-Kit⁺ myeloid progenitors (red), LKS (black), megakaryocyte-erythroid progenitors (blue), common myeloid progenitors (pink), GMPs (orange), ST-HSC (violet), and LT-HSC (green). Numbers represent average percentage of the distinct populations in BM of analyzed animals. See supplemental Figure 2A for complete quantification. (E) Number of cells in the distinct BM populations. At least 5 animals were included in each group. Graph shows 1 representative biological experiment of 3. (F) Representative flow cytometry plots from WT and dnTCF4 PB and their quantification. Left panel illustrates Ly6G (y-axes) and Ly6C (x-axes) expression in CD11b⁺ cells. Black boxes and numbers indicate the percentage of Ly6G⁺ Ly6C⁺ cells in CD11b⁺ population. Right graph indicates the percentage of Ly6C⁺ Ly6G⁺ in CD11b⁺ cells in PB of WT and dnTCF4 mice. At least 14 mice were included in each group. (G) Number of neutrophils per liter (L) in PB based on auto hematology analyzer. At least 18 mice were included in each group. (H) Percentage of neutrophils in BM in WT and dnTCF4 mice. Y-axis indicates the percentage of CD11b⁺ Ly6G⁺ Ly6C⁺ cells in BM; n = 6. (I) Number of CD11b⁺ Ly6G⁺ Ly6C⁺ cells in WT and dnTCF4 BM. Y-axis indicates the number of cells per leg. At least 8 mice were included per group. All animals included in Figure 1 were 12 to 15 weeks old, each represented by a dot symbol in panels E-I. Data indicate mean ± SD, and 2-tailed Student t test was used to assess statistical significance (*P < .05, **P < .01, ***P < .001, and ****P < .0001).