Eight-week-old male wildtype C57Bl/6 mice (Jackson Laboratory) were used for analysis of miRNA expression changes BMS-907351 chemical structure after 2/3 PH. Liver samples were obtained at 0, 1.5, 6, and 18 hours after surgery. Five mice were analyzed for each timepoint. Total RNA was isolated using Trizol and purified with the miRNeasy mini kit (Qiagen). miRNA expression profiling including labeling, hybridization, scanning, normalization, and data analysis was performed at Exiqon. Profiling was conducted with total RNA with RNA integrity number (RIN) values close to 8 measured with an Agilent

2100 Bioanalyzer. One μg total RNA of each sample and a common reference pool were labeled with Hy3 and Hy5 fluorescent label, respectively, using the miRCURY LNA Array power labeling kit (Exiqon). Hy3-labeled samples and Hy5-labeled common reference pool were mixed pairwise and hybridized to miRCURY LNA arrays v. 9.2 (Exiqon), which have a 61% coverage of the mouse miRNAs listed in miRBase v. 14.0. Hybridization was performed using a Tecan HS4800 hybridization

station. Slides were scanned using an Agilent G2565BA Microarray Scanner System and image analysis was carried out with ImaGene 7.0 software (BioDiscovery). Background correction was performed to remove nonbiological contributions to the measured signal.14 Quantified signals were normalized using the global Lowess (locally weighted scatterplot smoothing) regression algorithm.15 Log2 transformed median Hy3 (sample)/Hy5 (common reference pool) ratios were calculated for each capture probe (present Z-VAD-FMK datasheet in four replicates on the array). A two-tailed Student’s t test was performed between the samples obtained at 0 hours and the samples obtained at 1.5, 6, and 18 hours after 2/3 PH. Clustering was performed on miRNAs corresponding to capture probes with log2 Hy3/Hy5 ratios which passed filtering criteria of P < 0.001. The heatmap

was generated using the Euclidean metric. The log2 median ratio values were standardized Mannose-binding protein-associated serine protease by subtracting the mean followed by dividing by the standard deviation.16 miRIDIAN miRNA Mimics or Hairpin Inhibitors (Dharmacon) were introduced into Hepa1,6 mouse hepatoma cells (ATCC) at a final concentration of 20, 40, or 80 nM. Five × 104 cells were plated in 24-well plates (Corning) and transfected using Lipofectamine 2000 (Invitrogen) 24 hours later. Hepa1,6 cells transfected with chemically modified double-stranded or single-stranded oligonucleotides based on the sequence of miR-67 from C. elegans (both Dharmacon) were used as controls for miRNA mimics or inhibitors, respectively. For luciferase assays, cells were cotransfected with 30 ng of the pMIR-REPORT vector (Ambion) modified to contain the B-cell translocation gene 2 (Btg2) or ornithine decarboxylase 1 (Odc1) 3′ untranslated region (UTR) and 30 ng of the pSV-β-Galactosidase Control Vector (Promega) to monitor transfection efficiencies.