AML cells express high levels of Trib2 protein and correlate with E2F family members mRNA expression. (A) Real-time analysis of Trib2 mRNA levels in U937 cells following transduction with pLKO.1 control (Ctrl) or pLKO.1 Trib2. (B) Cell growth analysis in U937 cells transduced with pLKO.1 ctrl or pLKO.1 Trib2 assessed by trypan blue exclusion. (C) Cell death analysis in U937 transduced with pLKO.1 ctrl or pLKO.1 Trib2 assessed by (left) Annexin V and (right) DAPI staining. (D) Cell cycle analysis in sorted U937 cells transduced with pLKO.1 ctrl or pLKO.1 Trib2. Data presented are mean ± SD of duplicate cultures and representative of 2 independent experiments. (E) Western blot analysis of Trib2 protein expression in (top left) myeloid leukemia lines (K562, U937, ML-1, and Kasumi-1), (middle left) normal cell lines (CV1665 and CV1939), and (bottom left) normal peripheral blood (PB and N1-5). (Top right) Real-time analysis of Trib2 mRNA levels in AML patient samples. (Bottom right) Western blot analysis of Trib2 and E2F1 protein expression in normal PB (N6), AML patient samples (denoted by their subtypes), ALL patient samples, and U937 cells. Actin is shown as a protein loading control. (F) Correlation analysis of Trib2 vs E2F1 in the top 95th and 96th percentiles of patient samples from the dataset GSE1159³⁴ (N = 14) and GSE14468³⁵  (N = 21). P < .05 and correlation coefficient (Pearson r, close to +1) show significant positive correlations.