15, 16 Therefore, in this
study, we used only female mice. Eight-week-old WT (C57BL/6J) and TGR5−/− female mice (on C57BL/6J background; Merck Research Laboratories, Kenilworth, NJ)17 were maintained in a pathogen-free animal facility under a standard 12-hour light-dark R428 cycle. Mice were fed a diet containing 10 mg of 23(S)-mCDCA/kg diet or standard rodent chow for 3 days. After that, mice were fasted overnight and then injected intraperitoneally (i.p.) with a single dose of LPS (20 mg/kg) or phosphate-buffered saline (PBS), followed by feeding water ad libitum. Six hours after the injection, mice were killed, and the liver was removed for further analysis. All procedures followed National Institutes of Health (Bethesda, MD) guidelines for the care and use of laboratory animals. Reagents and plasmids are outlined in the Supporting Information. Cell culture and transient transfection are outlined in Supporting Information. Mouse bone-marrow–derived macrophages were derived according to previously published methods.18 Mouse Kupffer cells were prepared as described previously.13 Cells were pretreated with 23(S)-mCDCA (10 μM). Eighteen hours after treatment, the cells were treated with LPS (1 ng/mL), then collected for RNA isolation after a 4-hour incubation. Analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the staining for liver sections, is described in the Supporting Information.
Total MK 1775 RNA isolation from cells and tissues and quantitative real-time polymerase chain reaction (qRT-PCR) are described in the Supporting Information. The method for enzyme-linked immunosorbent assay (ELISA) is described in the Supporting Information. Protein extract preparation and immunoblotting analysis are described in the Supporting Information. HepG2 cells or mouse macrophages were transfected with p65 expression plasmid or control plasmid with or without
cotransfection of TGR5 plasmid. After 24-hour transfection, cells were treated with 10 μM of 23(S)-mCDCA or dimethyl sulfoxide (DMSO) (control) for 24 hours. Finally, nuclear proteins were extracted for electrophoretic mobility-shift assay (EMSA). EMSA assays were performed as previously described.19 The following oligonucleotide was used for the EMSA assay: NF-κB-binding site; 5‘- tcgagggctggggattccccat-3’. The plasmids, pFLAG-IκBα, pHA-β-arrestin2, and mTGR5, Fenbendazole were cotransfected into HEK293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cell treatment and immunoprecipitation for cells and liver tissue were performed as described in the Supporting Information. β-arrestin2 siRNA and control small interfering (si)RNA were purchased from Santa Cruz Biotechnology (Santa Cruz, California) and transfected into HepG2 cells using siRNA transfection reagent (Santa Cruz Biotechnology). Cell treatment is described in the Supporting Information. All data represent at least three independent experiments and are expressed as the mean ± standard deviation.