Briefly, 10 ��g of tissue or cell lysate or 25 ��l of conditioned medium were boiled for 10 min under reducing conditions and extracts separated on a 10% SDS-polyacrylamide gel, blotted onto nitrocellulose, and stained with 0.5% Ponceau S to assure equal protein loading and transfer. Membranes were blocked with 5% powdered milk and incubated overnight at 4��C with rabbit selleckchem anti-rat MMP-9 (1:1,000, AB 19016; Chemicon, Temecula, CA), followed by the appropriate alkaline phosphatase-conjugated secondary antibody for 2 h (Promega, Madison, WI). Immunodetected proteins were visualized utilizing nitroblue tetrazolium and bromo-chloro-indolyl-phosphate. Standardization was achieved by loading equal amounts of protein (tissue and cell lysates) or using equal cell numbers (cell lysates and conditioned medium).

Cell Isolation and Culture Cell lines. Mouse macrophage cell lines RAW 264.7 (ATCC no. TIB-71) and J774A.1 (ATCC no. TIB-67), human hepatoma cells Huh-7, the rat HSC line CFSC-2G (18) (kindly provided by Dr. M. Rojkind, Washington, DC), and the mouse cholangiocyte cell line 603B (19) were cultured under standard conditions as previously described (32, 33, 35) at 95% air-5% CO2 in a humidified atmosphere. Rat peritoneal macrophages. Rat peritoneal macrophages (pM��) were isolated using a standard protocol (13). Briefly, adult male Sprague-Dawley rat (250 g) were euthanized in a CO2 chamber and injected intraperitoneally through a 20-gauge needle with 50 ml of cold Ca2+, Mg2+-free PBS, followed by aspiration of macrophage-containing medium using the same syringe and centrifugation for 10 min at 400 g.

All procedures were done at 4��C and under sterile conditions. Macrophage yields were 1.4 �� 107 to 2.3 �� 107 cells/rat at a purity of 85% or higher as determined by phagocytosis of 3 ��m latex beads as previously described (35). Apoptosis was induced in vitro in 603B cholangiocytes by a single UV irradiation (200 mJ, Bio-Rad GS Gene Linker UV-chamber; Bio-Rad, Hercules, CA). Apoptotic cells were collected after 3 h, centrifuged at 13,000 g for 5 min, and resuspended in serum-free (1% penicillin/streptomycin; 4 �� 106 cells/ml) before each experiment. Apoptotic cell death was confirmed as previously established (9), assessing cellular criteria (cell rounding, cytoplasmatic membrane blebbing), nuclear morphology (condensed and fragmented nuclei by DAPI staining), and annexin V positivity by fluorescence-activated cell sorting.

It was routinely 80% or higher. For apoptotic cell engulfment experiments, 1�� 104 to 4 �� 104 apoptotic 603B cholangiocytes were added to unstimulated macrophages in serum-free Entinostat medium (freshly isolated rat pM�� or RAW cells, 3 �� 104 cells/well) for 1�C16 h. Determination of ECM-degrading Activities Determination of net interstitial collagenase and gelatinase activities.