Reduced G-CSF-R signaling in dnTCF4 HSPCs. (A) Histogram plots indicate levels of Stat3 phosphorylation (p-Stat3) in WT and dnTCF4 cells (LKS on left; c-Kit⁺ cells on the right) upon G-CSF stimulation. Gray histogram represents background signal (fluorescence minus one control [FMO]). Analysis was performed at time point t = 0, t = 2, and t = 5 minutes. (B) Quantification of panel A; n = 4. (C) Colony-forming assay in MethoCult M3231 supplemented with G-CSF and SCF using WT and dnTCF4 BM cells. A total of 1 × 10⁴ BM cells was plated per well. The y-axis indicates total number of CFU per 1 × 10⁴ BM cells; n = 5 (D) Percentage of c-Kit⁺ immature cells in WT and dnTCF4 G-CSF/SCF semisolid cultures assessed by flow cytometric analysis; n = 3. (E) Flow cytometric analysis of Ly6C⁺ Ly6G⁺ (gated on CD11b⁺) in cells from WT and dnTCF4 G-CSF/SCF semisolid cultures. The y-axis indicates the percentage of mature neutrophils; n = 5. (F) Representative histogram plot. The x-axis shows CD11b expression in cells from WT and dnTCF4 G-CSF/SCF semisolid cultures. (G) Quantification of panel F; n = 3. Data represent mean ± SD; 2-tailed Student t test was used to assess statistical significance, *P < .05, **P < .01, ****P < .0001.