Extracts were prepared from snap-frozen liver by homogenization in lysis buffer

(Tris-HCl 50 mM, NaCl 150 mM, ethylenediaminetetraacetic acid 1 mM, 1% Triton X-100, 0.5% Tween-20, and 0.1% sodium dodecyl sulphate), containing a protease-inhibitor cocktail (Roche), followed by centrifugation at 14,000×g for 15 minutes at 4°C. Supernatants were collected and activated with acetic acid/urea before analysis. Transforming growth factor β (TGFβ1) content of liver protein extracts were measured using a mouse TGFβ1 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN). Plates were read using the Bio-Rad (Hercules, INCB018424 concentration CA) microplate reader at 450 nm (with a 540-nm reference filter), and TGFβ1 concentrations were calculated from the standard

curve by the plate-reader software. Immortalized human HSCs (LX-2 cells; a gift from Dr. Scott Friedman) were seeded for 3 days into six-well plates at a density of 1 × 105 cells per well in M199 medium (Gibco, Grand Island, NY) with 5% fetal calf serum. Media were changed at day 3, and human PAR-1 agonist hexapeptide SFLLRN-NH2 (Sigma-Aldrich) and/or human PAR-2 agonist hexapeptide SLIGKV (Sigma-Aldrich) were added at varying concentrations. A scrambled hexapeptide (Auspep, Melbourne, Victoria, Australia) was used as a control. A further dose of either agonist or scrambled peptide was added at 24 and 48 hours, and culture medium and cells were harvested after 72 hours of peptide exposure. The collagen content of the cell-culture supernatant was measured using mafosfamide the Sircol Sirius red PLX4032 dye colorimetric assay (Biocolor, Newtown Abbey, Northern Ireland), as previously described,11 and TGFβ1 content was measured by ELISA. LX-2 cells were seeded onto 96-well plates at a density of 1 × 104 per well in 5% FCS/M199 media and

cultured overnight. The PAR-2 agonist peptide, SLIGKV, was added at concentrations from 0 to 100 μM at 24 and 48 hours. Human platelet-derived growth factor (PDGF)-BB (R&D Systems, Minneapolis, MN) was used as a positive control at a concentration of 25 ng/mL. Proliferation of activated HSCs was assessed using a colorimetric bromodeoxyuridine ELISA (Roche), according to the manufacturer’s instructions. Data are expressed as mean ± standard error of the mean. Statistical significance was determined by one-way analysis of variance with the Newman-Keuls post-test for multiple comparisons or the Student’s t test for comparisons between two groups, as appropriate, using GraphPad Prism 5.03 for Windows (GraphPad Software, Inc., La Jolla, CA). WT mice developed significant hepatic collagen deposition in response to CCl4 administration (Fig. 1A). No fibrosis was observed in WT mice given olive oil alone (data not shown). Quantitative analysis of histological fibrosis by computer-assisted morphometry in CCl4-treated WT mice showed marked fibrosis at 5 weeks (1.97% ± 0.