All liver sections were scored by two board-certified pathologists who were blinded to the identity of the samples. Lobular

necrosis was evaluated in liver sections stained with hematoxylin-eosin.25 Lobular necrosis selleck inhibitor was scored as follows: −, 0 foci; +/−, <2 foci; +, 2-4 foci; ++, >4 foci.25 Sections were examined in a coded fashion by BX-51 light microscopy (Olympus, Tokyo, Japan) equipped with a camera. We measured (1) the percentage of cholangiocyte apoptosis by semiquantitative terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling kit (Apoptag; Chemicon International, Inc.); (2) cholangiocyte proliferation by evaluation of the percentage of small and large cholangiocytes positive Opaganib cell line for PCNA5; and (3) intrahepatic bile duct mass (IBDM)5 of small (<15 μm)1 and large (>15 μm)1 bile ducts. IBDM was measured as the area occupied by cytokeratin-19–positive bile

duct/total area × 100. Proliferation was evaluated by immunoblots20 for PCNA in protein (10 μg) from lysate from spleen (positive control) and large cholangiocytes from WT and SR−/− BDL mice. Blots were normalized by β-actin.5 The intensity of the bands was determined by way of scanning video densitometry using the Storm 860 and the ImageQuant TL software version 2003.02 (GE Healthcare, Little Chalfont, Buckinghamshire, England). These experiments were performed in large cholangiocytes from WT and knockout 7-day BDL mice, a period where a marked ductal hyperplasia is observed.2, 12 We evaluated basal and secretin-stimulated cAMP levels (a functional parameter of cholangiocyte growth)13, 18 by commercially available RIA kits20; and phosphorylation of ERK1/2 by immunoblots in protein (10 μg) from cholangiocyte lysate. The intensities of the bands were determined by scanning video densitometry using a phospho-imager. Our small (negative control) and large cholangiocytes8 were treated at 37°C with 0.2% bovine serum albumin (BSA) (basal) or secretin (100 nM) for 48

hours in the absence or presence of preincubation (1 hour) with H89 (protein kinase A [PKA] MCE inhibitor, 30 μM) or PD98059 (mitogen-activated protein kinase kinase [MEK] inhibitor, 10 nM) before evaluating proliferation by CellTiter 96 Cell Proliferation Assay20 (Promega Corp., Madison, WI). Absorbance was measured at 490 nm on a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Data were expressed as the fold change of treated cells compared with vehicle-treated controls. In separate experiments, large cholangiocytes were treated with 0.2% BSA (basal) or secretin (100 nM) for 6 hours in the absence or presence of H89 (30 μM) or PD98059 (10 nM) before evaluating PCNA expression by way of immunoblotting,5 PKA activity,20 and phosphorylation of ERK1/2 by way of immunoblotting.5 The intensity of the bands was determined as described above.