Trib1 represses the neutrophil (Neu) program ex vivo. (A) Schematic of ex vivo eosinophil differentiation assay. Frequency of live cells (B) and day 10 cell output (C) using Trib1^+/+ or Trib1^ΔHSC BM. Frequency: n = 5 mice per group, pooled from 3 experiments; cell output: n = 3 wells per group, representative of 4 experiments. Frequency of live (D) cells and day 10 cell output (E) using Trib1^+/+ or Trib1^ΔEos BM. Frequency: n = 3 mice per group, representative of 6 experiments; cell output: n = 3 wells per group, representative of 5 experiments. (F) Representative histogram of Ly6G expression on SiglecF⁺CCR3⁺ eosinophils from Trib1^+/+ (blue) or Trib1^ΔHSC (red) IL-5 cultures. (G) Frequency of Ly6G expression on SiglecF⁺CCR3⁺ cells at day 10; n = 3 mice per group, representative of 4 experiments. (H) Representative histogram of Ly6G expression on SiglecF⁺CCR3⁺ eosinophils from Trib1^+/+ (blue) or Trib1^ΔEos (red) IL-5 cultures. (I) Frequency of Ly6G expression on SiglecF⁺CCR3⁺ cells; n = 3 mice per group, representative of 6 experiments. Eosinophils gated CD11b⁺SiglecF⁺CCR3⁺; neutrophils gated CD11b⁺Ly6G⁺SiglecF⁻. (J) Representative plots of day 10 IL-5 cultures of sorted GMPs from Trib1^+/+ and Trib1^ΔHSC mice, gated on live, CD11b⁺ cells. (K) Quantification of Ly6G expression on SiglecF⁺CCR3⁺ cells at day 10 of IL-5 GMP culture; n = 3-5 wells per genotype, representative of 2 experiments. (L) Quantification of granulocyte output at day 10 IL-5 culture of sorted GMPs; n = 3-5 wells per genotype, representative of 2 experiments. qPCR analysis of sorted neutrophils (CD11b⁺Ly6G⁺SiglecF⁻), and Ly6G⁻ and Ly6G⁺ eosinophils (CD11b⁺SiglecF⁺CCR3⁺) from day 10 IL-5 culture of Trib1^+/+ and Trib1^ΔHSC BM for Trib1 (M), Epx (N), and Ltf (O), relative to 18s, normalized to Trib1^+/+ neutrophils; n = 3, representative of 2 experiments. **P < .0084; ***P = .0002; ****P < .0001; unpaired Student t test. Frequencies and error bars are mean ± SEM of live cells. SCF, stem cell factor.