RUNX1-ETO repressed the RUNX3 P1 promoter through 2 conserved RUNX binding sites. (A) Schematic diagram of the RUNX3 P1 promoter showing the consensus RUNX, E-box, and C/EBP binding sites along with the promoter deletions analyzed in subsequent panels. (B) Wild-type and mutant RUNX3 P1 promoter constructs were cotransfected with pCMV-RUNX1-ETO and pRL-CMV into HeLa and U937 cells. The -1256-Luc was also cotransfected with pCMV-RUNX1-ETOΔ469 to examine the effect of C-terminal deletion on RUNX3 repression. (C) The -199-Luc promoter construct was cotransfected with 1 μg of pCMV-RUNX1-ETO or increasing amounts (1 μg, 2 μg, and 4 μg) of pCMV-RUNX1 together with pRL-CMV into HeLa cells. In all experiments, cotransfection with the same amount of empty pCMV was done in parallel. Transfection efficiency was normalized according to the cotransfected pRL-CMV Renilla luciferase activity. Results are presented as fold of repression by comparing the normalized firefly luciferase activity of the construct cotransfected with the expression plasmids to that cotransfected with empty pCMV. Results are expressed as mean plus or minus SE from triplicate assays. pCMV-RE indicates pCMV-RUNX1-ETO.