Hh signaling was also assessed in primary hepatocytes isolated from another 6 adult male mice 24

or 48 hours after sham surgery (n = 2 mice) or PH (n = 4 mice). Standard in situ liver perfusion and density gradient centrifugation techniques were used to isolate hepatocytes.18 Cells were processed immediately for immunocytochemistry or plated onto plastic dishes in 10% serum-supplemented Dulbecco’s modified Eagle medium (Sigma, St. Louis, MO) overnight. Medium containing nonadhered cells was removed the following morning, plates were washed with fresh medium, and adherent cells were harvested for analysis. To assess direct effects of Hh pathway inhibition on cell viability and proliferation, hepatocytes

were BAY 57-1293 supplier similarly isolated from four additional mice 24 hours after sham surgery (n = 2) or PH (n = 2) and cultured with either vehicle or cyclopamine (5 μM) in the presence of BrdU for 24 hours. Formalin-fixed paraffin-embedded livers and hepatocyte cytospins were prepared selleck chemicals as previously described.16 A detailed protocol and antibodies used are listed in Supporting Materials and Methods. Quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR) was performed using established protocols15; details are provided in Supporting Materials and Methods and Supporting Table 1. Proteins were isolated from whole liver tissue or primary hepatocytes. After quantification,

equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blot analysis was performed. A detailed list of antibodies is given in Supporting Materials and Methods. Statistical analyses were performed using GraphPad Prism v5.02 for Windows (GraphPad Software, San Diego, CA). Results of gene expression are grouped into pre-replicative (0-36 hours post-PH), replicative (36–100 hours post-PH), and post-replicative (100–216 hours post-PH) sets, and compared with 0-hour (quiescent) samples. Statistical significance was determined using one-way medchemexpress analysis of variance (ANOVA) and Bonferroni’s multiple comparisons test. Where mice were treated with either vehicle or cyclopamine, results were compared with respective vehicle-treated or cyclopamine-treated, 0-hour (quiescent) samples. Statistical significance was determined using unpaired, two-tailed Student t test. Significance was accepted at the 5% level, *P < 0.05, **P < 0.01, or ***P < 0.001. Hepatic expression of messenger RNAs (mRNAs) that encode Hh-pathway inhibitors (for example, Hh interacting protein [Hip]), Hh ligands (Indian Hh [Ihh], and sonic Hh [Shh]), the receptors that repress (patched [Ptc]) or promote (smoothened [Smo]) Hh signaling, and Hh-inducible transcription factors (glioblastoma [Gli]1 and Gli2) were evaluated at several distinct time points after PH.