Figure 7

Effects of overexpression of GATA-1/GATA-2 or AML1-ETO on the ETO promoter reporter. (A) HEL/MEG-01 cells were co-transfected with 15 μg ETO -729 to -259 bp promoter plasmid and 0 to 10 μg of GATA-1 plasmid. The luciferase activity is normalized against the ETO -729 to -259 promoter. The ETO promoter is activated by overexpression of GATA-1 in a dose-dependent manner. Similar results were obtained by co-transfection of HEL cells (data not shown). (B) MEG-01 cells were co-transfected with 15 μg ETO -729 to -259 bp promoter plasmid and 0 to 1 μg of AML1-ETO plasmid. The pGL3/basic and pGL3/SV40-promoter are used as negative and positive control, respectively. The luciferase activity is normalized against ETO -729 to -259 bp promoter. The ETO promoter is strongly repressed in a dose-dependent manner by AML-ETO. Western blotting shows exogeneous AML1-ETO (detected with anti-MTG) and endogeneous ETO expression (detected with anti-ETO). These experiments were repeated three times with similar results. Firefly was normalized to Renilla luciferase as internal control for transfection efficiency and the results are given as adjusted Relative Luciferase Units (AdjRLU). ***, p < 0.0001