CBFβ-MYH11 cooperated with RUNX1 to repress the RUNX3 P1 promoter through the same conserved RUNX binding sites. (A) The -1256-Luc promoter construct was cotransfected with pCMV-CBFβ-MYH11 or empty pCMV together with pRL-CMV into HeLa and U937 cells. (B) Wild-type and mutant RUNX3 P1 promoter constructs were cotransfected with the indicated amount of pCMV-CBFβ-MYH11 and/or pCMV-RUNX1 together with pRL-CMV into U937 cells. Cotransfection with the same amount of empty pCMV was done in parallel. In all experiments, transfection efficiency was normalized according to the cotransfected pRL-CMV Renilla luciferase activity. Results are presented as relative promoter activity by comparing the normalized firefly luciferase activity of the construct to that of pGL3-Basic or 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-CM indicates pCMV-CBFβ-MYH11.