Cell cultures, transient transfections, cell induction and luciferase assays

Human colon adenocarcinoma grade II cell line, HT29 (Sigma, Milan, Italy), was maintained in Dulbecco’s Modified Eagle’s Medium (Sigma) supplemented with 10% (v/v) heat-inactivated fetal bovine serum at 37℃ in a humidified 5% CO₂-containing atmosphere. Cell cultures were kept sub-confluent and transiently transfected for luciferase assays.

Transfection of HT29 was performed with Lipofectamine 2000 (Invitrogen Inc, USA). In brief, the day before transfection, cells were plated into Falcon 12 well plates at a density of 1.5 × 10⁵/ml in Optimem medium (Invitrogen Inc, USA). Cells were transiently transfected with 0.25 µg of each construct. To normalize the luciferase assay, 0.025 µg of the pRL-CMV vector (Promega) coding for the Renilla luciferase was transiently co-transfected. The pGL4-null was used as a negative control, whereas the pCMVluc (0.05 µg) was the positive control for the assay. After transfection, cells were treated for four hours with a mixture containing bacterial lipopolysaccharide (LPS) (10 µg/ml) (Sigma), interferon gamma (IFNγ) (100 units/ml) (R&D Systems, Minneapolis, MN, USA) and tumor necrosis factor alpha (TNFα) (2 ng/ml) (Sigma) for iNOS induction. Cell extracts were prepared 24 hours after induction, and 40 µl of lysate was used for the determination of luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) on a 20/20 ⁿ luminometer (Turner Biosystems, Sunnyvale, CA, USA), according to the manufacturers’ protocols.