RUNX1-ETO and CBFβ-MYH11 inhibited endogenous RUNX3 expression. (A) U937 cells were nucleofected with 20 μg of pCMV, pCMV-RUNX1-ETO, or pCMV-CBFβ-MYH11. Expression of fusion products was validated by RT-PCR (upper panel), and RUNX3 mRNA levels were determined by real-time RT-PCR and normalized using GAPDH (lower panel) 24 hours after nucleofection. Results are presented as relative RUNX3/GAPDH level by comparing the normalized RUNX3 level transfected with pCMV-RUNX1-ETO or pCMV-CBFβ-MYH11 to that transfected with empty pCMV. (B) Immunoblot analysis of RUNX1 and β-actin (loading control) expression in U937, A549, and HeLa cells. (C) A549 cells were transfected with 10 μg of pCMV, pCMV-RUNX1-ETO, or pCMV-CBFβ-MYH11 using lipofectamine 2000. The transfection efficiency from 3 independent experiments was 53% plus or minus 4%. Validation of fusion product expression (top panel) and measurement of RUNX3 mRNA levels (bottom panel) were done as described in panel A 48 hours after transfection. (D) RUNX3 P1 promoter construct -1256-Luc was cotransfected with pCMV-RUNX1-ETO, pCMV-CBFβ-MYH11, pCMV-CBFβ-MYH11 and pCMV-RUNX1 (0.5 μg each), or empty pCMV together with pRL-CMV into A549 cells. 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. Results are expressed as mean plus or minus SE from triplicate assays.