Inhibition of Sp1 binding attenuates hormone activation of the VEGF core promoter. (A) 22Rv1 cells were treated with DMSO or 0.1μM Mithramycin A for 24 hours. Sp1 and VEGF expression were measured by SYBR Green qRT-PCR using primers specific for Sp1 and VEGF and normalized to GAPDH. Values represent fold change relative to cells treated with DMSO vehicle control. (B) Schematic diagram of the 88bp region of the VEGF core promoter (as shown in Figure 5). Gray X’s indicate locations of two mutations created at the Sp1.2 and Sp1.3 binding sites, as described in the text. 22Rv1 cells were transfected with wildtype V88 or dual mutant (mSp1.2/.3) constructs followed by treatment with 0nM or 5nM R1881 as described above. (C) The Sp1.4 binding site in the V88 reporter construct was mutated (shown as above) and 22Rv1 cells were transfected as described for (B). (D) Sp1.4 binding site was mutated in the V2274 reporter construct and transfected into 22Rv1 cells treated with 0 or 5nM R1881 for 48 hours. Luciferase assays were performed as described previously. Luciferase activity is shown relative to average normalized luciferase activity in the absence of hormone. Experiments were repeated at least twice in triplicate. Significance was determined by Student’s t-test (* p<0.05, ** p<0.01, and *** p<0.001).