02 ml/g/ mouse) for the first 4 weeks and then once a week for the next 4 weeks. At week 4, 6 or 8, the mice were sacrificed. Partial livers were fixed, embedded in paraffin, and processed Carfilzomib for histology. Serial liver sections were stained with hematoxylin-eosin, Azan staining, Silver (Ag) staining, and Elastica van Gieson (EVG) staining, respectively. Total RNA from mice liver tissue was prepared as described previously. All animal procedures concerning the analysis of liver injury were performed in following the guidelines of the Kyoto University Animal Research Committee and were approved by the Ethical Committee of the Faculty of Medicine, Kyoto University. Cell lines and Cell preparation The human stellate cell lines LX-2, was provided by Scott L. Friedman.
LX-2 cells, which viable in serum free media and have high transfectability, were established from human HSC lines [26]. LX-2 cells were maintained in D-MEM (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum, plated in 60 mm diameter dishes and cultured to 70% confluence. Huh-7 and Hela cells were also maintained in D-MEM with 10% fetal bovine serum. HuS-E/2 immortalized hepatocytes were cultured as described previously [27]. LX-2 cells were then cultured in D-MEM without serum with 0.2% BSA for 48 hours prior to TGF��1 (Sigma-Aldrich, Suffolk, UK) treatment (2.5 ng/ml for 20 hours). Control cells were cultured in D-MEM without fetal bovine serum. miRNA transfection LX-2 cells were plated in 6-well plates the day before transfection and grown to 70% confluence.
Cells were transfected with 50 pmol of Silencer? negative control siRNA (Ambion) or double-stranded mature miRNA (Hokkaido System Science, Sapporo, Japan) using lipofectamine RNAiMAX (Invitrogen). Cells were harvested 2 days after transfection. Real-time qPCR cDNA was synthesized using the Transcriptor High Fiderity cDNA synthesis Kit (Roche, Basel, Switzerland). Total RNA (2 ��g) in 10.4 ��l of nuclease free water was added to 1 ��l of 50mM random hexamer. The denaturing reaction was performed for 10min at 65��C. The denatured RNA mixture was added to 4 ��l of 5�� reverse transcriptase buffer, 2 ��l of 10 mM dNTP, 0.5 ��l of 40U/��l RNase inhibitor, and 1.1 ��l of reverse transcriptase (FastStart Universal SYBR Green Master (Roche) in a total volume of 20 ��l. The reaction ran for 30 min at 50��C (cDNA synthesis), and five min at 85��C (enzyme denaturation).
All reactions were run in triplicate. Chromo 4 detector (BIO-RAD, Hercules, CA, USA) was used to detect mRNA expression. The primer sequences are follows; MMP13 s; 5��-gaggctccgagaaatgcagt-3��, as; 5��-atgccatcgtgaagtctggt-3��, TIMP1 s; 5��-cttggcttctgcactgatgg-3��, as; 5��-acgctggtataaggtggtct-3��, Drug_discovery ��1-procollagen s; 5��-aacatgaccaaaaaccaaaagtg-3��, as; 5��-cattgtttcctgtgtcttctgg-3��, and ��-actin s; 5��-ccactggcatcgtgatggac-3��, as; 5��-tcattgccaatggtgatgacct-3��.