HepG2 human hepatoma cells (HB-8065; ATCC, Manassas, VA) were cultured in MEM (ATCC) with 10% FBS (Invitrogen, Carlsbad, CA), 4 mM glutamine (Thermo Scientific Hyclone, Waltham, MA) and 1× Penicillin and Streptomycin (Thermo Scientific Hyclone) on cell bind surface plates (Corning Inc, Corning, NY) at 37 °C. For transfection assays, 3 × 105 cells were seeded per well in 12-well plates. 24 h after seeding, cells were transfected in complete media with 500 ng of test DNA, along with 15 ng of pCMV β-galactosidase plasmid (Clontech, Mountain View, CA) and 485 ng of pUC19 DNA, using 2 µl of Fugene HD (Roche, Indianapolis, IN) per 1 µg of DNA. Cells were harvested 30 h after addition of DNA by scraping into ice-cold 1× PBS, pelleted by centrifugation and suspended in 100 µl of 1× Reporter lysis buffer (Promega, Madison, WI). Cell extract was prepared by repeated freeze-thawing; 5 µl of the extract was used for each assay. Luciferase assays were carried out using the Luciferase assay system (Promega, Madison, WI), with activity measured on a Spectromax LS (Molecular devices, Sunnyvale, CA). β-galactosidase assays were carried out using the Galacto-Light System (Tropix, Benford, MA).
