HepG2 cells were grown in six-well plates (Corning, NY, USA) in MEM with 10% fetal bovine serum (FBS; Life Technologies, Rockville, MD, USA) and 2 mm glutamine. Cells (3.6 × 105 cells per well) were washed with 2 ml of medium without FBS and then transfected using Fugene 6 (Roche Diagnostics, Indianapolis, IN, USA) with a total of 3.3 µg DNA (2 µg test plasmid, 0.13 µg pCMV-β-galactosidase as an internal control and 1.2 µg pUC19 DNA) for 5 h. Complete medium (2 ml) containing FBS was then added and incubation was continued for 25 h. Cells were washed with 1× phosphate-buffered saline and resuspended in 100 µl of 1× reporter lysis buffer (Promega, Madison, WI, USA). Cells were lysed by three freeze–thaw cycles, followed by vortex mixing for 15 s. Twenty microliters of each lysate was assayed for luciferase activity using the Luciferase Assay System (Promega), with activity measured for 60 s on an Lmax Plate Luminometer (Molecular Devices, Sunnyvale, CA, USA). Five microliters of each lysate was assayed for β-galactosidase activity for 30 s using the Galacto-Light™ Plus System (Applied Biosystems, Benford, MA, USA) and the Lmax Plate Luminometer.
