Fig. 3.

Luciferase assay of SLC22A5 enhancer/promoter luciferase constructs. a Effect of E2 on the reporter activity of different lengths of SLC22A5 promoter luciferase constructs. Hormone-depleted MCF-7 cells were transfected with different lengths of SLC22A5 promoter luciferase plasmids for 16 h. Cells were then treated with vehicle/E2 for 6 h and lysed for luciferase analysis. b Schematic representation of SLC22A5 gene structure in the genomic context, showing the ER binding site (chr5:131741886-131742949, +8544 to +9607 from the transcriptional start site), visualized using the UCSC genome browser (http://genome.ucsc.edu). c Effect of E2 on the reporter activity of the SLC22A5 promoter luciferase construct with or without the intronic +9 kb enhancer. Luciferase analysis was performed as described above. d Potential transcription factor binding sites within the intronic +9 kb enhancer, showing the predicted half ERE, AP-1 site, full ERE, and CRE/NR4A2 site. e Effect of E2 on the reporter activity of SLC22A5 promoter-enhancer luciferase constructs, each carrying a deletion or mutation within one of the predicted binding sites. Luciferase analysis was performed as described before