Dysregulation of TLR4 signaling pathway in Runx1 KO neutrophils occurs prior to the committed neutrophil stage. (A) Representative FACS plots of intracellular TNF-α production by neutrophils (CD11b⁺Ly6G⁺) after stimulation of whole BM with vehicle or 100 ng/mL LPS for 4 hours. Runx1 PMN KO = Runx1^f/f; MRP8-Cre. (B) Quantification of the frequency of TNF-α⁺ neutrophils and relative MFI of the TNF-α⁺ neutrophils normalized to control neutrophils run in the same experiment (n = 4 from 4 experiments, mean ± SD, 1-way analysis of variance and Tukey’s multiple comparisons test). (C) Presence of the expected wild-type, floxed (f), or deleted Runx1 polymerase chain reaction products from tail clips (with a small amount of contaminating blood) and FACS-purified neutrophils (CD11b⁺SiglecF⁻F4/80⁻Ly6G⁺). (D) Western blot showing total RUNX1 levels in FACS-purified neutrophils and splenocytes from the same mice. ***P ≤ .001; ****P ≤ .0001. ns, not significant.