19 (Supporting Information Materials and Methods). To quantify liver fibrosis, 5-μm sections were stained with Picrosirus red (Sigma, St. Louis, MO) and counterstained with fast PD0325901 concentration green (Sigma, St. Louis, MO). Collagen stained with Sirius red was quantitated in the sections that were randomly chosen (under ×20 magnification, 10 fields each from sample) as described.20 To localize and characterize cells that produce and/or respond to Hh ligands and OPN, formalin-fixed,

paraffin-embedded livers were prepared for immunohistochemical analysis as described.6, 7 Protocols and antibodies used are listed in Supporting Information Materials and Methods and Supporting Information Table 1. Real-time quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and western immunoblot analysis were performed using established protocols.7 Details are www.selleckchem.com/products/PD-98059.html provided in the Supporting Information Materials and Methods and Supporting Information Table 2. Hepatic stellate cells (HSCs) were isolated from normal Sprague-Dawley rats as described21 (Supporting Information Materials and Methods). A similar isolation/culture protocol was

used for studies involving mouse primary HSCs.6 Day 4 HSCs were used in all experiments. The human HSC line LX-2 was cultured in serum-supplemented Dulbecco’s modified Eagle’s medium. Primary human HSCs were isolated as described.21 To evaluate the effects of Hh signaling on HSCs, day 4 HSC cultures were grown for an additional 24 hours in medium containing either exogenous Hh agonist (SAG) at a concentration of 0.3 μM, 5 μM cyclopamine (Toronto Research Chemicals, Inc., Toronto, ON, Canada), an inhibitor of Hh signaling, or 5 μM tomatidine (Calbiochem, San Diego, CA), a catalytically inactive analog of cyclopamine5, 21 (tomatidine serves as a control for cyclopamine). In separate experiments,

recombinant OPN (rOPN) or vehicle was added to cultures to assess their effects on HSC activation. A 100-ng/mL dose was used in this study selleck screening library because it stimulated the greatest effects in vitro.22 The effects of inactivating OPN were subsequently assessed by treating HSCs with the human OPN RNA aptamer OPN-R3 or its biologically inactive mutant, OPN-R3-2 (both synthesized by Dharmacon, Lafayette, CO).23 Aptamers (100 nmol/L) were added to medium for 48 hours before harvest. This concentration of OPN aptamer has been shown to inhibit adhesion, migration, and invasion in the breast cancer cell line MDA-MB-231 (which highly expresses OPN and is a standard tool for evaluating OPN actions).23 All cell experiments were performed at least in duplicate. Total RNA and protein were harvested before treatment and at the end of treatment and were analyzed by way of QRT-PCR and immunoblotting, respectively.