Recombinant TG2 protein (rTG2) was used as a reference in Western blot analysis. A fragment of 1·5 Kb long of the TG2 promoter region from Caco-2 cells was cloned in a firefly luciferase reporter vector pGL3 (Promega). Caco-2 cells

were plated in a 24-well plate and transfected transiently with TG2 promoter construction together with a Renilla luciferase vector. Transient transfection was carried out using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 4·105 per well in a six-well plate. When cells reached 40–50% confluence they were washed with 2 ml Opti-MEM (Invitrogen) and incubated with a DNA-Lipofectamine Enzalutamide manufacturer (Invitrogen) mixture for 6 h in a humidified 5% CO2 environment. After 6 h of incubation the transfection medium was replaced

with fresh culture medium, and cells were incubated for 24 h. Subsequently, cells were incubated with cytokines alone (TNF-α 10 ng/ml, IFN-γ 200 UI/ml) or with the addition of inhibitors of signalling pathways (SP600125 20 µM, Ly294002 2 µM, sulphasalazine 10 µM) for another 24 h. Luciferase activity was measured in cellular lysates using the Dual-Luciferase Reporter Assay System Kit (Promega), according to the manufacturer’s protocol. For each sample firefly luciferase data were normalized to the Renilla luciferase internal control. Relative luciferase units (RLU) are referred to the non-stimulated control. To evaluate the expression of surface TG2, THP-1 cells were treated for 20 h with TNF-α 10 ng/ml and IFN-γ Epigenetic Reader Domain inhibitor 200 UI/ml, and inhibitors of signalling pathways. Cells were incubated with the anti-TG2 monoclonal antibodies (4E1G9, 2G3H8, 5G7G6 or 1H7H9) produced in our laboratory [16]. Then cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Ig)G (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Flow

cytometry analysis was performed in a fluorescence Methane monooxygenase activated cell sorter (FACS)Calibur flow cytometer (BD Biosciences), and data were analysed using FlowJo software (Tree Stars, Ashland, OR, USA). To investigate whether the proinflammatory cytokines TNF-α, IFN-γ, IL-15, IL-6 and IL-1 modulate TG2 expression, we measured TG2 mRNA levels by quantitative (q)RT–PCR in five human cell lines from different cell lineages (Caco-2 and HT29, intestinal epithelia; A549 and CALU-6, lung epithelia; THP-1, monocyte-like) stimulated for 24 h with the cytokines mentioned. In all cell lines tested, except for A549, IFN-γ was the most potent inducer of TG2 expression (Fig. 1). The highest induction of TG2 by IFN-γ was observed in THP-1 cells (fold increase = 20·2). In the two intestinal epithelial cell lines, Caco-2 and HT29, the up-regulation of TG2 transcript by IFN-γ was about 18-fold while TG2 levels were increased by 6·9- and 7·3-fold in A549 and CALU-6, respectively.