Int J Colorectal Dis (2008) 23:305–317 TTT CAG-3′ and β-actin (Invitrogen GmbH) forward 5′-CTG GCA CCC AGC ACA ATG-3′, reverse 5′-CCG ATC CAC ACG GAG TAC TTG-3′ in an ABI Prism 7000 sequence detection system. PCR reaction was set up with Sybr® Green PCR Master mix (Invitrogen) containing 0.3 μmol/l primers each and 1 μl of RT-product in 25 μl volume. A two-step amplification protocol was chosen consisting of initial dena- turation at 95°C for 10 min followed by 45 cycles with 15 s denaturation at 95°C and 30 s annealing/extension at 60°C. Finally, a dissociation protocol was performed to control specificity of amplification products. Relative expression of CXCL10 was then calculated using the comparative threshold-cycle (CT) method. The amount of target mRNA in each sample was normalized to the amount of β-actin mRNA designated as calibrator, to give ΔCT (CT β- actin− CT CXCL10). The relative expression of CXCL10 was calculated as the CXCL10=β actin ratio ¼ 2$CT. MRNA expression is presented as fold increase calculated in relation to unstimulated cells after normalization to β-actin. Transient transfections Caco-2, HT29 and DLD1 cells were plated in 24-well plates, 24 h before transfection (Costar, Corning, NY), at a density of 50,000/well in 1 ml medium. After 24 h, the cells were transfected with 250 ng of respective plasmids along with 20 ng of renilla plasmid using the non-liposomal formulation FuGENE (Roche Molecular Biochemicals, Mannheim, Germany). On the day of transfection, fresh medium was added. FuGENE was added to the plasmid DNA at a ratio of 3 μl/μg DNA. FuGENE was prediluted in 100 μl serum-free medium and added drop-wise to the concentrated plasmid DNA. After 15–25 min at room temperature, the mixture was added to the cells. The plasmids TGL-IP10, TGL-IP10 κB2 Mut were kind gifts from Dr. Richard M. Ranshoff (Cleveland, OH) and were described elsewhere [26]. Luciferase assays Twenty-four hours after transfection, cells were stimulated with IL-1β (0.1,1, 2, 10 and 50 ng/ml), TNF-α (2, 10 and 50 ng/ml), IFN-γ (2, 10 and 50 ng/ml), IL-1β (1 ng/ml) + TNFα (50 ng/ml), IL-1β (1 ng/ml) + IFN-γ (50 ng/ml) and TNFα (50 ng/ml) + IFN-γ (50 ng/ml) for 24 h, harvested and lysed, and luciferase was assayed by using a luciferase kit (Promega). Values were normalised to renilla luciferase. Enzyme-linked immunosorbent assay To study the synergistic effects of cytokines on CXCL10 secretion, supernatants were collected at 0, 3, 6, 12 and 24 h in Caco-2 and HT29 cells stimulated with IL-1β (1 ng/ml), 307 TNF-α (50 ng/ml), IFN-γ (50 ng/ml), IL-1β (0.5 ng/ml) + TNFα (50 ng/ml), IL-1β (0.5 ng/ml) + IFN-γ (50 ng/ml) and TNFα (50 ng/ml) + IFN-γ (50 ng/ml) and stored at −70°C until measured. CXCL10 protein secretion was measured by ELISA according to the manufacturer’s protocol. Preparation of nuclear extract Caco-2 cells were grown for 24 h, pre-treated for 1 h with BAY 11-7082 (10 μM) and then stimulated with respective cytokines for 30 min. Cells were washed with 10 ml cold phosphate-buffered saline (PBS) twice, harvested, centrifuged at 1,000×g for 5 min and resuspended in 1 ml of cold PBS. Centrifuged briefly, excess PBS was removed, and the pellet was resuspended in ice-cold extraction buffer (20 mM hydroxyethyl piperazineethanesulfonic acid [HEPES] pH 7.8, 10 mM KCL, 0.1 mM ethylenediamine tetraacetic acid [EDTA], 1 mM dithiothreitol and 0.5 mM phenyl- methanesulphonylfluoride [PMSF]) by gentle pippeting. Cells were then kept on ice for 15 min, and 30 μl of 10% Nonidet NP-40 was added and vortexed briefly for 10 s. The lysed cell suspension was centrifuged for 30 s, and the supernatant was discarded. The pellet was resuspended in 50 μl extraction buffer containing 400 mM NaCl, agitated vigorously for 15 min at 4°C. Then, the cell suspension was centrifuged for 5 min at 4°C, and the supernatant containing the nuclear extract was collected and stored at −80°C until further use. Electrophoretic mobility shift assay and super-shift assay For binding reactions, 5 μg of nuclear extracts were incubated in 20 μl reaction mixture containing 40 mM HEPES pH 7.5, 50 mM NaCl, 1 mM EDTA, 1mM DithitreitolHSCl, 1 mM PMSF, 1.28 μg/ml pBluescript vector DNA as half of the reaction and the rest being nuclear extract plus other components. Oligonucleotide duplex probes (Promega) were end-labelled with T4 polynucleotide kinase and [γ32P] adenosine triphosphate; 1× 104 cpm were then added to the reaction mixtures and incubated overnight at 4°C. Reaction products were analysed by non-denaturing electrophoresis in a 5% polycralamide gel with 0.5× Tris– borate–EDTA buffer at room temperature. Gels were then exposed directly to X-ray film at −70°C for autoradiography. For competition, unlabelled oligonucleotides were added in molar excess at room temperature for 15 min before adding radiolabelled probe. In super shift experiments, nuclear extracts were incubated with anti-p65 anti-body (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Statistical analysis Statistical analyses were carried out using the Prism software packet (Version 3.0, Graphpad Software, San