The pellet was finally re-suspended in TS buffer supplemented with 5 mM dithiothreitol and used immediately
(on ice at all times) by addition of [3H]dexamethasone with or without excess unlabelled dexamethasone. After overnight incubation on ice, free ligand was removed by charcoal dextran adsorption Selleck ZVADFMK and bound radioligand determined in supernatants by liquid MCC950 solubility dmso scintillation, essentially as previously described [9–11]. Quantitative RT-PCR Quantitative transcript expression was examined after reverse transcription (using a Taqman reverse transcription kit (Applied Biosystems)) using a PE Applied Biosystems ABI7700 and PE Applied Biosystems Gene Expression Assay™, incorporating sequence-specific forward, reverse and fluorescently labelled probes (see Table 2). Table 2 qRT-PCR kits used in these studies. Transcript mRNA Applied Biosystems Primer Kit Human 18S rRNA Hs99999901_sl TGF-β1 Hs00171257_ml TIMP1 Hs00171558_ml COL1A1 Hs00164004_ml Rat GAPDH 4352338E COL1A1 Rn00801649_g1 In vivo animal study and tissue analysis Rats were administered CCl4 mixed 1:1 (v/v) with olive oil – 2 ml/kg body weight by i.p. injection
– twice weekly for 8 weeks to generate liver fibrosis . Control animals were administered olive oil alone (1 ml/kg body weight). Once a week between CCl4 treatment, rats received 4A3COOHmethyl (20 mg/kg body weight). After 8 weeks, animals were killed by cervical dislocation and samples of various tissues removed and fixed in 10% formalin in PBS. Blood was allowed to clot prior to centrifugation S3I-201 to obtain serum for analyses. Serum ALT levels, α-smooth muscle
actin immunostaining and sirius red staining were performed as previously outlined [6, 46]. Acknowledgements This project was supported by the Wellcome Trust and a aminophylline Proof of Concept Grant from Scottish Enterprise. Electronic supplementary material Additional file 1: Supplemental table S1. Competition of substituted progestins for binding to rat liver microsomes (DOC 99 KB) Additional file 2: Supplemental table 2. Competition of dexamethasone derivatives for binding to rat liver microsomes. (DOC 42 KB) References 1. Wallace K, Burt AD, Wright MC: Liver fibrosis. Biochem J 2008, 411:1–18.CrossRefPubMed 2. Fallowfield JA, Mizuno M, Kendall TJ, Constandinou CM, Benyon RC, Duffield JS, Iredale JP: Scar-associated macrophages are a major source of hepatic matrix metalloproteinase-13 and facilitate the resolution of murine hepatic fibrosis. J Immunol 2007, 178:5288–5295.PubMed 3. Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterström RH, Perlmann T, Lehmann JM: An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 1998, 92:73–82.CrossRefPubMed 4.