αGalCer, alpha-galactosylceramide; α-SMA, alpha-smooth muscle actin; ctgf, connective tissue growth factor; FACS, fluorescent activated cell sorting; FFPE, formalin-fixed paraffin-embedded;

Foxf1, Forkhead box F1; Gli, glioblastoma; Hh, Hedgehog; HSC, hepatic stellate cells; iNKT, invariant natural killer T; LMNC, liver mononuclear cell; MCD, selleck chemicals methionine choline deficient; mmp9, matrix metalloproteinase 9; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NK, natural killer; NKT, natural killer T; Ptc+/−, patched; Shh, sonic hedgehog; Tgf-β, transforming growth factor beta; Vcam1, vascular cell adhesion molecule 1. C57BL/6 wildtype (WT) (Jackson Laboratories, Bar Harbor, ME), Patched-deficient (Ptc+/−) mice (from R.J. Wechsler-Reya, Duke University, NC), and CD1d-deficient mice (from Z.P. Li, Johns Hopkins University, Baltimore, MD) were fed a methionine-choline deficient (MCD) diet or control chow for 8 weeks. Ptc+/−

mice have only one copy of patched, a Hedgehog (Hh)-pathway repressor. Therefore, they are unable to silence Hh-signaling and exhibit excessive Hh-pathway activity.25 NKT cells are genetically absent in CD1d-deficient mice.26 Formalin-fixed paraffin-embedded (FFPE)-livers were analyzed27 (for detailed protocol and antibodies, see Supporting Information Materials and Methods). Total liver RNA extraction

and messenger RNA (mRNA) quantification by real-time qualitative reverse-transcription polymerase chain reaction (qRT-PCR) were performed as detailed in Supporting Information Angiogenesis inhibitor Materials and Methods.27 Primers are in Supporting Information Table 1. Hydroxylproline content in whole liver specimens was quantified colorimetrically.27 LMNC were isolated from WT mice28, 29 medchemexpress and characterized by fluorescent antibody cell sorting (FACS) as detailed in Supporting Information Materials and Methods. LMNC were cultured in complete NKT media (RPMI 1640, supplemented with IL2 [10 ng/mL; Biolegend] and 10% heat-inactivated fetal bovine serum),28 with or without the NKT cell ligand, alpha-galactosylceramide (αGalCer; 100 ng/mL; Axxora, Cat. no. 306027, CA), for 24 hours. This αGalCer dose elicits maximal iNKT activation.30 Conditioned media were then added to primary murine HSCs for 1 day and RNA was harvested for qRT-PCR. Experiments were performed in duplicate wells and repeated twice. Murine cholangiocyte 603B line (from Yoshiyuki Ueno, Tohoku University, Sendai, Japan, and G. Gores, Mayo Clinic, Rochester, MN),31 rat HSC line 8B (from M. Rojkind, George Washington University, Washington, DC),32 and murine invariant hybridoma cell line DN32 (from Albert Bendelac, University of Chicago, Chicago, IL)33 were cultured according to established protocols.