Trib1 expression induces an aggressive AML phenotype. (A) Morphologies of Hoxa9-expressing AML cells with (hi) or without (null) Trib1 expression (right). Reverse transcription (RT) PCR of Trib1 expression in hi cells (left). Scale bars, 20 μm. (B) Fluorescence-activated cell sorting analysis shows decreased expression of Mac1 and Gr-1 (left) and an increased CD34-positive fraction (right) in Trib1 hi cells. The numbers indicate frequencies (%) of Mac1/Gr-1-double-positive, Mac1-single-positive, and CD34-positive fractions. (C) Decreased expression of the p42 isoform of C/EBPα in Trib1 hi cells. (D) Enhanced and prolonged phosphorylation of ERK1/2 in Trib1 hi cells. (E) Increased proliferation of Trib1 hi cells. ***P < .001. (F) Increased 5-ethynyl-2′-deoxyuridine (EdU) incorporation in Trib1 hi cells. (G) Gene set enrichment analysis shows correlation of the cell cycle pathway (left) and inverse correlation of the C/EBPα network gene sets (right). FDR, false discovery rate; NES, normalized enrichment score; p-val, P value. (H) AML developed by transplantation of Trib1 hi cells with a median survival time of 97.1 days, whereas transplantation of Trib1 null cells failed to show AML development in vivo. Significance between 2 cohorts was examined by a log-rank test.