Increased TNF-α production by Runx1 KO neutrophils in response to TLR4 stimulation. (A) Representative FACS plots of intracellular TNF-α production by monocytes (CD11b⁺Ly6G⁻) after stimulation of whole BM with vehicle or 100 ng/mL LPS for 4 hours. (B) Quantification of the frequency of TNF-α⁺ monocytes and relative MFI of TNF-α in the TNF-α⁺ monocytes normalized to control monocytes run in the same experiment (n = 6 from 4 experiments). (C) 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. (D) Quantification of the frequency of TNF-α⁺ neutrophils and relative MFI of TNF-α in the TNF-α⁺ neutrophils normalized to control neutrophils run in the same experiment (n = 6 from 4 experiments). (B,D) Bar graphs depict independent data points with the mean ± SD; 2-tailed unpaired Student t tests. (E) Quantification of the frequency of TNF-α⁺ neutrophils (CD11b⁺Ly6G⁺) after stimulation of whole BM for 4 hours with TLR agonists (n = 4 to 6, as indicated from 6 experiments). Bar graphs depict independent data points with the mean ± SD. Statistics represent the results of a 1-way analysis of variance followed by Sidak’s multiple comparison test to compare the means of the control and Runx1 KO samples for each TLR agonist. (F-H) Absolute quantification by CBA of inflammatory factor levels in the supernatant of 200 000 FACS-purified neutrophils (CD11b⁺SiglecF⁻F4/80⁻Ly6G⁺) stimulated for 8 hours with vehicle or 100 ng/mL LPS. (I) Quantification by CBA of TNF in the supernatant of 200 000 purified monocytes stimulated for 8 hours with vehicle or 100 ng/mL LPS. (F-I) Bar graphs depict independent data points with the mean ± SD. Five replicates from 3 experiments were performed for each condition with all results above the limit of detection (blue arrowhead) plotted. Statistics represent 2-tailed unpaired Student t tests. **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.