5), containing 1 mM of EDTA, 2 mM of MgCl2, 50 mM of KCl, 1 mM of dithiothreitol, and protease inhibitors by incubating at 4°C for 30 minutes and centrifuging at 14,000 rpm for 5 minutes. A detailed protocol and antibodies used are described in Supporting Materials and Methods. Liver sections were stained for

5′-bromo-2′-deoxyuridine (BrdU)-positive nuclei with the BrdU labeling and detection kit (Roche, Indianapolis, IN), according to manufacturer’s instructions. Ten randomly selected high-power fields (40×) of liver Torin 1 sections from 4-6 mice per group were analyzed. The number of BrdU-positive cells were counted and expressed as a percentage of the total number of cells, as visualized by hematoxylin-eosin staining. For evaluation of eNOS gene expression, RNA was isolated from liver tissues harvested at 0.5-72 hours post-PH using the Qiagen RNeasy minikit, according to the manufacturer’s

instructions (Qiagen Sciences, Germantown, MD). Reverse transcription (RT) was performed using 2 μg of total RNA in a first-strand complementary DNA (cDNA) synthesis reaction with the high-capacity cDNA RT kit (Applied Biosystems, Foster city, CA), as recommended by the manufacturer. The cDNA product was amplified by quantitative RT polymerase chain reaction (qRT-PCR) in an ABI prism 7700 sequence-detection system buy GDC-0199 (Applied Biosystems) with primers specific for mouse eNOS and cyclophilin, as described previously.15 Hepatocytes were isolated from 6-8-week-old WT and eNOS−/− male mice by the two-step collagenase perfusion protocol, as described previously, Celecoxib with modifications optimized for

mice.16 For hepatocyte proliferation assays, cell preparations with viability over 95%, as screened by Trypan Blue exclusion assay, were seeded at a low density of 200,000 cells/35 mm of Primaria tissue-culture wells (BD Labware, Franklin Lakes, NJ) in Williams E complete media, with additives for 3 hours to ensure hepatocyte adherence to plates. Subsequently, hepatocytes were maintained in Williams E minimal media free from serum and growth factors for 20 hours before treatment with growth factors. A detailed protocol of doses and duration of EGF treatment of hepatocytes in vitro and pretreatment of cell-signaling-pathway–specific inhibitors, to assess the role of eNOS in EGF-mediated mitogenic signaling and proliferation, is described in Supporting Materials and Methods. Data are represented as the mean ± standard deviation (SD) of at least three independent experiments. The statistical significance of difference between groups was analyzed by the unpaired Student’s t-test. Values of P < 0.05 were considered statistically significant. Activation of MAPKs and immediate early genes are hallmarks of early events activated within minutes of PH.