Figure 4. (A and C) Schematic representation of the human miR200s promoters on chromosome 1 (A) and on chromosome 12 (C) cloned in pGL3 basic reporter vector in which site specific mutagenesis was performed to mutate the 2 most distal MBS. The core MBS of each site is reported with the distance from transcription start site (TSS). The mutated nucleotides in each mutant are underlined. (B and D) luciferase assays were performed in HEK293 cells co-transfecting the wt and mutant reporter vectors with the pcDNA3HA (empty vector, white bars) or the pcDNA3-c-mybHA (c-myb expressing, black bars) expression vectors. Transfection efficiency was normalized by co-transfecting a renilla luciferase-expressing vector. Assays were performed 48 h after transfection. Fold-activation of each reporter vector co-transfected with pcDNA3HA was arbitrarily set to 1. Luciferase assays data are reported ± standard deviation. Statistical significance was calculated by two-tailed Student t test. ** p ≤ 0.01. n.s. = not significant. Experiments were performed twice in triplicate.