Enhancer reporter assay

SNPs rs4631830 and rs13394027 were selected for enhancer reporter assay. The SNP-centered DNA fragment (~500 bp) was amplified from human genomic DNA and cloned into upstream of the SV40 promoter in the pGL3 promoter vector (Promega, Madison, WI). For allele-specific enhancer activity, site-directed mutagenesis (Stratagene, La Jolla, CA) was used to create allele-specific construct. The oligo sequences used in this mutagenesis were listed in Supplementary Table S6. In addition, the PSA (prostate-specific antigen) enhancer-containing fragment (~600 bp) was used as a positive control. Plasmid DNAs were prepared and purified using QIAprep Miniprep kit (Qiagen,Valencia,CA). For plasmid transfection on white 96-well tissue culture plates, 100 ul suspension (3 × 10⁵ cells/ml) of LNCaP cells per well were applied to reverse transfection with luciferase reporter plasmids together with pGL75 using X-tremeGENE HP DNA Transfection Reagent (Roche Applied Science, Penzberg, Upper Bavaria, Germany) following the manufacturer’s instructions. 48 hours later, cells were analyzed for luciferase activities using Dual-Glo® Luciferase Assay System (Promega). All data came from five replicate wells.