α-SMA, alpha-smooth muscle actin; BDL, bile-duct ligation; CCl4, carbon tetrachloride; DTT, dithiothreitol; ECL, enhanced chemiluminescense; EDTA, ethylenediamine tetraacetic acid; ECM, extracellular matrix; EGTA, ethylene glycol tetraacetic acid; ERK, extracellular signal-related protein kinase; FBS, fetal bovine serum; H&E, hematoxylin and eosin; HSC, hepatic stellate cells; HRP, horseradish peroxidase; IL-1α, interleukin-1 alpha; JAK, Janus kinase; LPS, lipopolysaccharide; mRNA, messenger RNA; MMP, matrix

metalloproteinase; NF-κB, nuclear factor kappa light-chain enhancer of activated B cells; PDGF, platelet-derived growth factor; RT-PCR, reverse-transcription AZD5363 nmr polymerase chain reaction; siRNA, short interfering RNA; TCA, trichloroacetic see more acid; TGF-β, transforming growth factor beta; TIMP-1, tissue inhibitor of metalloproteinase-1; TNF, tumor necrosis factor; TNFR, TNF-receptor; TNFR-DKO, TNFR1/R2 double knockout. Wild-type, TNFR1 knockout mice, TNFR2 knockout mice, and TNFR-DKO mice (10-18 weeks old) (C57BL/6 strain), a generous gift of Dr. Bluethmann (Discovery Technologies, Hoffmann-La Roche Ltd., Basel, Switzerland), were obtained by the propagation

of homozygous pairs. The animals had free access to water and standard purified rodent diet throughout the study. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals published

by the National Institutes of Health (Bethesda, MD). HSCs were isolated by perfusion with collagenase and cultured as described.21 In addition to primary mouse HSCs, we used the human HSC cell line, LX2. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) plus 10% Clomifene fetal bovine serum (FBS), and antibiotics were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were serum-starved with 0.5% FBS before using TNF-α, PDGF-BB, interleukin-1 alpha (IL-1α), and IL-1β (PreproTech EC, London, UK) or lipopolysaccharide (LPS) (from Escherichia coli serotype 0128:B12; Sigma-Aldrich Quimica SA, Madrid, Spain). Neutralizing antibodies against human TNFR1 and TNFR2 (R&D Systems, Minneapolis, MN) were used at a concentration of 10 μg/mL. In vitro short interfering RNA (siRNA) transfection was performed using commercially available siRNA (Santa Cruz Biotechnology, Heidelberg, Germany) as previously described.21 Unless otherwise stated, all reagents were from Sigma-Aldrich. Total RNA from HSCs, mouse tissue, or LX2 cells was isolated with TRIzol reagent (Invitrogen, Paisley, UK). Real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed with the iScript One-Step reverse transcription (RT)-PCR kit with SYBR Green (Bio-Rad Laboratories SA, Madrid, Spain). Primer sequences were designed based on published sequences (Table 1).