Figure 6. (A)HEK293 cells transfected with pcDNA3-ZEB1HA expression vector or empty vector pcDNA3HA were tested by western blot for ZEB1HA expression 72 h after transfection using an anti-HA antibody. β-actin expression was used for normalization.(B)Luciferase assays were performed in HEK293 and MCF-7 cells co-transfected with the reporter vectors containing the miR200 promoter on chromosome 1 (left histograms) or the miR200 promoter on chromosome 12 (right histograms), and the expression vector pcDNA3-ZEB1HA or the empty vector pcDNA3HA.(C)Luciferase assays were performed in HEK293 (left histograms) and in MCF-7 cells (right histograms) stably transfected with the expression vector pcDNA3-c-mybHA or the empty vector pcDNA3HA. These cells were co-transfected with reporter vectors containing the miR200 promoter on chromosome 1 (top histograms) or the miR200 promoter on chromosome 12 (bottom histograms) and increasing amounts (from 0–50 ng/sample) of the expression vector pcDNA3-ZEB1HA. Fold-activation of cells co-transfected with the reporter vectors and the empty vector pcDNA3HA (white bars) was arbitrarily set to 1. Luciferase assays data are reported ± standard deviation. Statistical significance was calculated by two-tailed Student t test. Experiments were performed twice in triplicate.