Polyclonal rabbit anti-Endo180 antibody was a gift from Dr. Clare M. Isacke (Breakthrough Breast Cancer Research Centre, The Institute of Cancer research, London, UK). This polyclonal antiserum was initially raised against purified human Endo180  and we have previously shown that it also reacts with rat Endo180 . Anti-desmin antibody (Cat. No. D-1033, mouse monoclonal), anti-α smooth muscle actin antibody (Cat. No. A-2547, mouse monoclonal), anti-glial fibrillary acidic protein antibody (Cat. No. G-2969, rabbit polyclonal), and anti-β-actin antibody (Cat. No. A-5316, mouse monoclonal) were obtained from Sigma. Secondary horseradish peroxidase-conjugated antibodies were obtained from GE Healthcare. Collagenase H, DNase I and protease inhibitors were obtained from Roche. Pronase E was obtained from MERCK. Culture media and Hank's balanced salt solution were from Gibco BRL (Invitrogen). Fetal bovine serum (Cat. No. A15-151) was obtained from PAA Laboratories GmbH. Antibiotics were obtained from BioWhittaker. Bovine serum albumin, collagen from calf skin (Cat. No. C-3511), and Optiprep (60% w/v) were obtained from Sigma. All other chemicals and reagents were obtained from Sigma unless otherwise specified.
Preparation and culture of hepatic stellate cells
Animals received human care according to the guidelines of University of Oslo. Male Wistar rats, weighing 650 to 800 g, were used in experiments. HSCs were prepared by in situ pronase/collagenase perfusion at 37°C and density gradient centrifugation as described  with some modifications. Briefly, the animal was anesthetized with pentobarbital and the liver was perfused via the portal vein for about 10 min with calcium-free/magnesium-free Hank's balanced salt solution (HBSS) without phenol red. The liver was then removed and perfused with 0.35% pronase in HBSS containing calcium and magnesium and phenol red for about 20 min. This was followed by perfusion in a re-circulating system with a mixture of 0.035% collagenase and 0.1% pronase in HBSS for 20 min. Thereafter, the digested liver was suspended in HBSS containing 0.015% DNase and filtered through a sterile 100 μm nylon mash. The filtered suspension was divided in four 50-ml centrifuge tubes and centrifuged at 540 × g at 14°C for 7 min. The pellets were then re-suspended in HBSS containing DNase and centrifuged as described above. This was repeated once and the pellets of non-parenchymal cells were pooled and re-suspended in HBSS. The cell suspension was then mixed with 29% Optiprep solution in HBSS (to give 11.6% Optiprep), divided in two 50-ml centrifuge tubes and then overlaid with 5 ml HBSS containing 1% BSA and centrifuged at 1400 × g at 14°C for 23 min in a Eppendorf centrifuge (5810 R), without brake. After centrifugation, the interface layer was collected and washed twice in HBSS. After washing, the cells were re-suspended in Dulbecco's Minimum Essential medium (DMEM) containing 2 mM L-glutamine, 25 mM HEPES, 4.5 g/L glucose, penicillin (100 U/ml), streptomycin (100 μg/ml), and 18% fetal bovine serum (FBS), and counted. The cell viability was over 95% as determined by the trypan blue staining. Cells were cultured on 10 cm dishes (Costar) at a density of 3 × 106 cells/dish (for extraction of total protein and total RNA) or 6-well culture plates (Costar) at a density of 2 × 105 cells/well (for endocytosis experiments). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. The following day, the cells were washed twice in HBSS to remove non-adherent cells and fresh growth medium containing 18% FBS was added. One day later (day 2), the cells were washed again as above and incubated with growth medium containing 10% FBS for the next 5 days.
In some experiments, the cells were activated for 8 days in 75 cm flasks, trypsinized, recovered by centrifugation and then sub-cultured (first passage) in 10 cm dishes or 6-well plates and used after 3 to 5 days.
Oil Red-O staining
Oil Red-O staining was used to detect cytoplasmic lipid droplets. Cells were washed with phosphate-buffered saline (PBS), pH 7.4, and fixed in 1% paraformaldehyde at room temperature for 60 min. The cells were then washed with 50% isopropanol and stained with 0.3% Oil Red-O for 10 min at room temperature, washed with PBS, and then the nuclei were stained with hematoxylin for 2 min. Finally, the cells were washed with PBS and fixed with 1% PFA before they were photographed. Microphotographs were taken using a light microscope (Leica DMIL) connected to a digital camera (Wetzler GmbH).
Labelling of denatured collagen
Radio-labelled tyramine cellobiose (125I-TC) was prepared by reacting TC with Na125I (Perkin-Elmer) in Iodogen tubes (Pierce) and was then coupled covalently to heat-denatured collagen (5 mg in 500 μl borate buffer, pH 9.4) after activating the 125I-TC with the cross-linking agent cyanuric chloride, essentially as described by Pittman et al . The labelled protein (125I-TC-collagen) was dialyzed against PBS using 10000 Da dialysis cassettes (Pierce Biotechnology) to remove non-coupled 125I-TC.
Uptake and degradation experiments
Cells were washed three times with HBSS and then 1 ml DMEM containing 1% bovine serum albumin (BSA) was added. After pre-incubation for 45 min at 37°C, cells were incubated with 125I-TC-collagen at 37°C. For experiments where inhibitors were used, cells were either treated with inhibitor or control amounts of vehicle (DMSO) during the last 15 min of the pre-incubation period. At the end of the incubation period, the medium was removed and the cells were washed three times with ice-cold PBS, scraped in lysis buffer (0.1% SDS, 0.1 N NaOH), transferred to tubes and then precipitated with trichloracetic acid and BSA at final concentrations of 10% and 0.5%, respectively. Cell lysates were kept on ice for 15 min and centrifuged to separate acid-soluble fraction from the acid-precipitable fraction. Cell-associated acid-soluble and acid-precipitable radioactivities were measured using a γ counter. It should be noted that the 125I-TC moiety formed after degradation of 125I-TC-collagen is not released to the medium but remains trapped in degradative compartments . Total uptake was calculated by subtracting radioactivity bound to the cell surface at 4°C from total cell-associated radioactivity measured at 37°C and expressed as the sum of acid-precipitable and acid-soluble radioactivity. Radioactivity bound to control wells (wells containing no cells) was less than 0.1% of the total radioactivity added to the medium. On each of days 1, 2, and 3 cells were counted directly in duplicate wells under an inverted microscope. On days 4, 5, 6, and 7 duplicate wells were trypsinized and cell number determined by use of a hemocytometer.
Western blot analysis
At different time points, cultured HSCs were washed three times with PBS, scraped, spun down, and cell pellets were frozen at -20°C until preparation of total protein. Total protein was prepared from frozen cell pellets by addition of sample-loading buffer (Tris buffer pH 6.8, 2% Sodium dodecyl sulfate (SDS), and 10% sucrose) containing protease inhibitors. In some experiments cells were washed and lysed directly in sample-loading buffer containing protease inhibitors and stored at -20°C. Cell lysates were kept on ice for 30 min with occasional vortexing, boiled for 7 min, centrifuged, and protein concentration in the clarified lysates was determined using the BCA Protein Assay kit (Pierce). Equal amounts of protein (30–50 μg) in cell lysates were separated by 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions and electrotransferred to a polyvinylidine difluoride membrane (Millipore). The membrane was blocked in PBS containing 0.1% (v/v) Tween-20 (PBS-Tween) and 5% skim milk for 1 h at room temperature and then blotted with rabbit anti-Endo180 polyclonal antibody for 1 h. After several washes with PBS-Tween, the membrane was incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG to detect bound anti-Endo180. The membrane was washed with PBS-Tween and secondary HRP-conjugated antibody was visualized by ECL detection system (GE Healthcare) and exposed to film. The membrane was stripped for re-blotting with β-actin and one or two marker proteins.
Reverse-transcriptase polymerase chain reaction (RT-PCR)
At different time points, cultured HSCs were washed three times with PBS, scraped, spun down, and cell pellets were frozen at -20°C until preparation of total RNA. Total RNA was extracted from frozen cell pellets using Versagene RNA Cell Kit (Gentra Systems) according to the manufacturer's instruction. One microgram total RNA was reverse transcribed using SuperScript II RNase H- Reverse Transcriptase (Invitrogen). After cDNA synthesis, the cDNA mixture was used as template for PCR. Routine PCR was performed using DyNAzyme (Finnzymes) and the primers (rat/mouse Endo180 forward 5'-CAC GGG AAG CCG TGT ACT AT-3' and reverse 5'-CCT CCA GGA CAG TGT GGA TT-3', human Endo180 forward 5'-GGA ACC CAA CAT CTT CCT CA-3' and reverse 5'-CCG GTC ACA CTC ATA CAT GC-3', and β-actin forward 5'-AGC CAT GTA CGT AGC CAT CC-3' and reverse 5'-TCT CAG CTG TGG TGG TGA AG-3') were designed by the Primer 3 Output program http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.
LX-2, a human HSC line, was provided by Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York). The murine M1-4 HSC line was provided by Dr. Wolfgang Mikulits (Institute of Cancer Research, Medical University of Vienna, Austria). The rat HSC lines, BSC and MFBY2, were provided by Dr. Hidekazu Tsukamoto (Keck School of Medicine, University of Southern California, Los Angeles) and Dr. Sato Kenzo (School of Life Science, Tottori University Faculty of Medicine, Japan), respectively. The murine and rat HSC lines were cultured in DMEM containing 10% FBS (Gibco BRL, Invitrogen) and kept at 37°C as described for primary cultures. LX-2 cell line was maintained under identical conditions but in Medium 199 instead of DMEM. For Western blotting and RT-PCR analyses, the cells were cultured to 80–90% confluency in 10 cm dishes and harvested as described for primary culture HSCs. For endocytosis, cells were cultured in 12-well plates at 1 × 105 cells per well twelve hours before the start of experiments and endocytosis was measured as described above. Duplicate wells were trypsinized and cell number of each cell culture was determined by a hemocytometer.