Materials and Methods

Isolation and culture of rat HSCs and chemicals
Primary HSCs were isolated from male Sprague-Dawley rats (200-250g) as we previously described 23. Passaged HSCs were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). Cultured HSCs were used at Passage #4-9. Human oxidized LDL was purchased from Intracel Company (Frederick, MD) and used before expiration. The presence of endotoxin in ox-LDL was < 0.1 units/ml, as measured by Limulus Amebocyte lysate assay kit (Whittaker M.A. Bioproducts, Walkersville, MD). Wnt3a was purchased from R & D Systems (Minneapolis, MN). PPARγ antagonist PD68235 was kindly provided by Pfizer (Ann Arbor, MI). Curcumin (purity > 94%) and κ-carrageenan were purchased from Sigma (St. Louis, MO). 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2), a natural PPARγ agonist, was a product of Cayman Chemical Company (Ann Arbor, MI).

Western blotting analyses
Protein samples were prepared from whole cell extracts, separated by 10% SDS-PAGE, and electro-blotted onto PVDF membrane. Target proteins were visualized by using the ECL method (Amersham, Piscataway, NJ). Mouse anti-LOX-1 monoclonal antibody was kindly provided by Dr. Tatsuya Sawamura (National Cardiovascular Center Research Institute, Japan). Other primary and secondary antibodies were presented in Table 1. Densities of bands in Western blotting analyses were normalized with the internal invariable control. Levels of target protein bands were densitometrically determined by using Quantity One® 4.4.1 (Bio-Rad, Hercules, CA). Variations in the density were expressed as fold changes compared to the control in the blot.

Immuno-staining
Serum-starved HSCs were treated with or without human ox-LDL (10 μg/ml) in the presence or absence of curcumin (20 μM) for 24 hr before the fixation with 4% paraformaldehyde for 15 min at room temperature (RT). After blocking with 5% BSA for 1 hr, fixed cells were incubated with anti-human ox-LDL antibody (1: 100) (Cat# AB3230, Chemicon Company, Billerica, MA) overnight at 4°C. After three washes with phosphate-buffered saline (PBS), sections on slides were incubated with goat anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (1:500) (Invitrogen, Carlsbad, CA) at RT for 1 hr. Sections incubated with secondary antibodies alone were used as negative controls (data not shown). DAPI (100ng/ml) (Molecular Probes Company, Eugene, Oregon) was added in the mounting solution for nuclear staining. Sections were viewed under Leica DM 400 B microscope (North Central Instruments, Maryland Heights, MO).

Preparation of nuclear protein extracts
Nuclear extracts were prepared as we previously described 28. In brief, after washing twice with PBS, cells were evenly re-suspended in Buffer A (10 mM HEPES-KOH pH7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM PMSF). After incubation on ice for 10 min, cells were mixed by vortex for 30 seconds and centrifuged at 8000g for 10 seconds. Pellets were re-suspended in Buffer C (20 mM HEPES-KOH pH7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF) and incubated on ice for 15 min before vortex. The lysates were centrifuged at 8000g at 4°C for 2 min, and the resulting supernatants were taken as nuclear protein extracts, and stored at -80°C until use.

Plasmids and transient transfection assays
The lox-1 promoter luciferase reporter plasmid (p-2336/+36-Luc), containing a 2336 bp fragment of the 5′-flanking promoter region of the LOX-1 gene, and some of the reporter plasmids with various lengths of the lox-1 promoter were kindly provided by Dr. Jawahar L. Mehta (Department of Internal Medicine, University of Arkansas for Medical Sciences) 29. Additional lox-1 promoter luciferase reporter constructs with shorter promoter regions were created by PCR and enzyme ligation using p-2336/+36-Luc as a template (see the following for details). The PDGF-βR promoter luciferase reporter plasmid pPDGF-βR-Luc (pβ12) was a gift from Dr. Keiko Funa (Ludwig Institute for Cancer Research, Uppsala, Sweden) 30. The type I TGF-β receptor promoter luciferase reporter plasmid pTβ-RI-Luc (pES1.0) was kindly provided by Dr Michael Centrella (Yale University, New Haven, CT, USA) 31. The PPARγ cDNA expression plasmid pPPARγ, containing a full size of PPARγ cDNA, was a gift from Dr. Reed Graves (Department of Medicine, University of Chicago). The dominant negative PPARγ expression construct pdn-PPARγ was a gift from Dr. Krishna V. Chatterjee 32. The Wnt signaling luciferase reporter plasmids TOPflash and its mutant FOPflash were kindly provided by Dr. Randall T. Moon (Department of Pharmacology, School of Medicine, University of Washington) 33. TOPflash contained 8 copies of TCF/LEF binding sites. FOPflash was a counterpart control for TOPflash with site-directed mutations in the TCF/LEF binding sites 33.
Transient transfection of semi-confluent HSCs in 6-well plates was performed using the LipofectAMINE® reagent (Invitrogen Corp.), as we previously described 23. Each treatment had triplicates in every experiment. Each experiment was repeated, at least, three times. Luciferase activity assays were conducted as we previously described 23. Transfection efficiency was determined by co-transfection of a β-galactosidase reporter, pSV-β-gal (Promega, Madison, WI). β-galactosidase assays were performed using an assay kit from Promega Corp, according to the manufacturer's instruction. Luciferase activities were expressed as relative unit after normalization with β-galactosidase activities. Results were combined from, at least, three independent experiments.

Generation of a LOX-1 cDNA expression plasmid
Total RNA was extracted from HSCs using Trizol reagent and reversely transcribed into cDNA, which was used as a template for PCR amplification of LOX-1 cDNA. The PCR primers were designed according to GenBank accession number: NM_138648, and use the following primers: (F) 5′-CCC AAG CTT ATG ACT TTT GAT GAC AAG ATG AAG C-3′; (R) 5′-GG GGT ACC CC CTA AAT TTG CAA ATG ATTT GTC TTC-3′. The primers were tailed with HindIII site (the forward primer) and KpnI site (the reverse primer). The PCR product was subcloned into pCDNA3.1/Zeo+ expression vector (Invitrogen Corp. Carlsbad, CA) at HindIII/KpnI sites. The subcloned LOX-1 cDNA was verified by DNA sequencing.

Promoter deletion and site-directed mutagenesis
The lox-1 promoter luciferase reporter plasmid p-2336/+36-Luc, also termed pLOX-1-Luc, was used as a PCR template to generate constructs with various lengths of the gene promoter. Primers in Table 2 were used for generating the plasmids by PCR. The primers were tailed with KpnI site (forward primers) or BglII site (reverse primer), and the PCR products were subcloned into pGL3-Basic (Promega) at KpnI/BglII sites. The site-directed mutagenesis was carried out using the GeneTailor Site-Directed Mutagenesis kit (Invitrogen), according to the manufacture's instruction. The sequence of 5′-GGC ACA TTT TTT ACA AAT GTA GTG TGA CTT ACT CTC TTT GAA TTT CAG TTT C-3′ (-310 to -258) containing the putative TCF/LEF-1 binding site in the plasmid p-2336/+36-Luc, was mutated to 5′-GGC ACA TTT TTT ACA AAT GTA GTG TGA CTT ACT CTC ATA GAA TTT CAG TTT C-3′ in p-2336/+36-Mut-Luc. The primers used for mutagenesis were in Table 3. The mutations were verified by DNA sequencing.

Real-time polymerase chain reactions (PCR)
Total RNA was extracted from cells using Trizol reagent (Invitrogen) following the manufacturer's instruction. Real-time PCR was carried out using SYBR green, as previously described 34. Total RNA was treated with DNase I prior to the synthesis of the first strand of cDNA. First-strand cDNA was synthesized using total RNA as templates and oligo-dT as primers. Samples were run on a Bio-Rad MyiQ™ real-time PCR machine (Bio-Rad). mRNA levels were expressed as fold changes after normalization with endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as suggested by Schmittgen et. al. 35. The primers for PCR were presented in Table 3.

Electrophoretic mobility shift assays (EMSA)
Synthesized single-stranded oligonucleotide probes were biotin-labeled using the Biotin 3′ End DNA Labeling Kit (Pierce Company, Rockford, IL), and annealed to form oligonucleotide duplex after labeling. EMSA were conducted using LightShift Chemiluminescent EMSA kit (Pierce Company, Rockford, IL), following the protocol provided by the manufacturer. Briefly, the annealed, biotin-labeled double-stranded oligonucleotide probes were incubated with 3 μg of nuclear extracts at room temperature for 30 min. The mixtures were subjected to 6% non-denatured polyacrylamide gel electrophoresis, and electroblotted onto nylon membrane. Proteins bound to the biotin end-labeled oligonucleotide probes were detected using the streptavidin-horseradish peroxidase conjugate and the chemiluminescent substrate. The following probes were used. P(TCFwt) contained a putative TCF-1 binding site: 5′-GTG TGA CTT ACT CTC TTT GAA TTT CAG TTT CTG TC-3′ (-289/-259). P(TCFmut) contained a mutated TCF-1 binding site: 5′-GTG TGA CTT ACT CTC ATA GAA TTT CAG TTT CTG TC-3′.

Statistical analyses
Differences between means were evaluated by an unpaired two-sided Student's t-test (P< 0.05 considered as significant). Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by ANOVA with the Dunnett's test for post hoc analysis.