Viability assays confirmed that these treatments did not significantly alter

selleckchem endothelial viability after 4 hours of treatment. WT mice and VAP-1–deficient mice (C57BL/6) expressing enzymatically active or inactive hVAP-1 on the endothelial cells under the control of the mouse tie 1 promoter have been described,24 and they were used to study the role of VAP-1 in MAdCAM-1 induction in vivo. All mice were handled in accordance with the institutional animal care policy of the University of Turku. MA [0.4% (wt/vol)] was administered in the drinking water of the animals (freshly made every day) for 14 days. After the mice were sacrificed, tissue samples from PPs and MLNs were excised and used for protein and RNA analysis. In order to study MAdCAM-1 induction in the intact

selleck products human liver, we used a Krumdieck tissue slicer (TCS Biologicals) to cut aseptic, 250-μm-thick slices of live liver tissue, which could be studied for up to 48 hours ex vivo. The liver tissue was incubated in Williams’ E media (Sigma) supplemented with 2% FBS, 0.1μM dexamethasone (Sigma), and 0.5μM insulin (Novo-Nordisk). Tissues were stimulated with MA (50 μM) and enzymatically active recombinant vascular adhesion protein 1 (rVAP-1) produced in Chinese hamster ovary cells (500 ng/mL; Biotie Therapies, Turku, Finland) before MAdCAM-1 protein and RNA analysis. The viability of the excised tissue slices was tested with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma) before and after the stimulation period (details are provided in the Supporting Information Materials

and Methods). Total RNA was extracted with the RNeasy mini Kit click here (Qiagen, United Kingdom) and analyzed as described in the Supporting Information Materials and Methods. MAdCAM-1 protein expression was determined by western blotting and immunoprecipitation techniques. The protocols and antibodies are described in the Supporting Information Materials and Methods. Multicolor fluorescence confocal microscopy was used to localize the expression of MAdCAM-1 in HECs. MAdCAM-1 expression in human liver tissue was investigated in formalin-fixed, sucrose-embedded tissues with NovaRED immunostaining. The presence of murine MAdCAM-1 in PPs and MLNs was examined by immunofluorescence. The protocols and antibodies are described in the Supporting Information Materials and Methods and Supporting Information Table 3. Formalin-fixed, sucrose-embedded sections (10 μm thick) were incubated with JY cells and PBLs from PSC patients (n = 3) for 30 minutes at room temperature. In certain experiments, tissue sections were incubated with an anti–MAdCAM-1 antibody (P1; 1 μg/mL; Pfizer), and JY cells and PBLs were blocked with anti-α4β7 [actin 1 (ACT-1); 1 μg/mL; a gift from M.