Transfection and Luciferase Assay.

Transfections were carried out using Lipofectamine 3000 (Invitrogen) for MCF-7 cells and ZR-751 cells and Lipofectamine 2000 (Invitrogen) for HMECs following the manufacturer’s instructions. Briefly, cells were allowed to reach 70% confluency at the time of transfection. Lipid–DNA complexes were prepared by adding DNA and Lipofectamine to serum-free medium and incubated at room temperature for 30 min. The cells were washed and the Lipid–DNA complexes were added. The amount of plasmid DNA was kept constant with empty vector in transient transfection experiments. After 6 h, the media volume was doubled with serum-free DMEM and supplemented with serum to attain a final concentration of 10% serum.

For luciferase assays, human PRAMEF2 promoter fragment (−1,650 to −850) was cloned into the pGL4.24 vector (Promega) to obtain PRAMEF2-luc reporter construct. The PRAMEF2^mut-luc2 reporter construct was generated by site-directed mutagenesis (Mutagenex). The cells were harvested 24 h posttransfection, and luciferase activity was determined using Dual Luciferase Reporter assay system (Promega) as per the manufacturer’s protocol. Briefly, cell extracts were prepared in 1X Passive Lysis Buffer, supplied by the manufacturer. In total, 20 µl cell extracts were added to 100 µl Luciferase Assay Reagent II, and Firefly luciferase activity was measured with a luminometer (Berthold systems). This signal was quenched by addition of 100 µl Stop and Glo reagent, followed by measuring the Renilla luciferase activity. The difference in transfection efficiency across samples was normalized by cotransfecting pRL-TK, which expresses Renilla luciferase. Error bars are mean ± SD of three independent experiments with duplicate samples.