RUNX1 and E2A-PBX1 coregulate a subset of genes and are both required for cell growth in E2A-PBX1–positive leukemic lines. (A) Immunoblot of E2A/E2A-PBX1 in the 697 line treated with shScr, shE2A-N, or shPBX1-C. Actin was used as the loading control. (B) Venn diagram showing the overlap of downregulated genes, relative to Scramble, in the 697 line treated with shE2A-N (#1), shPBX1-C (#1), or shRUNX1 (#1). (C) Heatmap of RNA-seq results for 2 experiments with 2 differentially designed shRNAs (#1 and #2) revealing a subset of genes that are downregulated in either the E2A-PBX1 (by shE2A-N or shPBX1-C) or RUNX1-depleted 697 line. The gene symbols and binding by E2A-PBX1 and RUNX1 are denoted on the right. (D) RT-qPCR assays showing the relative mRNA levels of indicated genes in the 697 cell line treated with Scramble, shE2A-N, shPBX1-C, or shRUNX1. Depletion of E2A-PBX1 and RUNX1 reduces cell growth (E) and colony formation (F). Cell growth and colony numbers were determined after the shRNA-mediated knockdown. Data are presented as relative to Scramble-treated cells. Statistics by Student t test. **P < .01; ***P < .001. (G) Images of representative colonies from indicated treatments in panel F. Scale bar, 0.5 mm.