Mutation of ARE sites attenuate hormone activation of the VEGF promoter. (A) Schematic diagram of a 2kb region of the VEGF promoter showing the ARE I-III binding sites. Thick X’s indicate sites that were mutated. (B) Shown are the wild type (WT) and mutant (MUT) sequences of the ARE I, ARE II, and ARE III binding sites. Site-directed mutagenesis was performed as described in text, using primers containing these mutated bases. (C) Wild type V2274 and mutant (mARE II) luciferase reporters were transfected into 22Rv1 cells treated with 0nM or 5nM R1881 for 48 hrs. Luciferase assays were performed as previously described. (D) Mutant (mARE III) and wild type V2274 were transfected into 22Rv1 cells as in (C). (E) Wild type and mutant V411 constructs were transfected into 22Rv1 cells as in C. (F) The mutant V411 construct was also transfected into LNCaP cells and treated as in C. Luciferase activity is shown relative to average normalized luciferase activity in the absence of hormone. Experiments were repeated three times in triplicate. Significance was determined by Student’s t-test (**p<0.01).