FOXA1-binding sites are DNA methylation- and cell-context-sensitive enhancers. (A) Reporter assays were performed using in vitro methylated or unmethylated enhancers cloned within a CpG-free luciferase reporter vector and transfected in MCF7 or LNCaP cells. “Empty” refers to the control reporter construct lacking an enhancer. Numbering of FOXA1-binding sites is consistent with Figure 1E. Results are means and standard deviations from a representative experiment performed in triplicates. (B) Reporter assays were performed using unmethylated reporter constructs that were transfected in MCF7 or LNCaP cells. Results are means and standard deviations from a representative experiment performed in triplicates. (C) Reporter assays were performed using unmethylated reporter constructs that were transfected in HEK293 cells. Results are means and standard deviations from a representative experiment performed in triplicates. (D) Reporter assays were performed using unmethylated reporter constructs that were transfected in MDA-control or MDA-FOXA1 cells, as indicated. The day before transfection, 2.5 μg/mL tetracycline was added to the cells. Results are means and standard deviations from a representative experiment performed in triplicates. For all experiments, luciferase activities are expressed relative to that obtained for the control reporter plasmid lacking an enhancer, which was set to 1.
