Cyp27a1

Liver or brain tissues were lysed with RIPA buffer containing protease inhibitor (PMSF) on ice for 30 min and centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was reserved and the protein concentrations were determined with BCA assay. An equal amount of protein was separated through SDS-PAGE running and then transferred to PVDF membranes (Millipore, Boston, MA, USA). The antibodies used were as follows: CYP27A1 1:5000 (Abcam, Boston, MA, USA), CYP27B1 1:1000 (Abcam, Boston, MA, USA), VDR 1:1000 (CST, Boston, MA, USA), β-actin 1:5000 (Abcam, Boston, MA, USA), and Goat anti-rabbit IgG 1:3000 (CST, Boston, MA, USA). Fluorchem FC 2 software was used to analyze the image and the relative expression of target proteins and normalized to β-actin protein.

Immunoblotting was performed as described previously16 (link). Protein isolated from whole livers was loaded onto 10% sodium dodecyl-sulphate polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were probed with anti-CYP7A1 (Santa Cruz Biotechnology, Dallas, TX), CYP27A1, SLC10A1, α-SMA (Abcam, Cambridge, UK) and phospho-JNK, JNK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Denvers, MA), followed by horseradish peroxidase-conjugated anti-mouse or rabbit IgG (Cell Signaling Technology). Immune complexes were visualized using enhanced chemiluminescence (GE Healthcare).

Frozen liver tissues were homogenized in buffer containing 0.25-M sucrose and 10-mM phosphate (pH 7.4). Nuclear fractions were extracted from parts of frozen liver samples using CelLytic^TM NuCLEAR^TM Extraction Kits (Sigma-Aldrich Japan, Tokyo, Japan). Samples were then subjected to 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to polyvinylidene difluoride membranes as described previously [23 (link)]. Membranes were then incubated overnight at 4°C with primary polyclonal antibodies against alpha smooth muscle actin (α-SMA), cytochrome (CYP)7A1, CYP27A1 (Abcam plc, Cambridge, UK), constitutive androstane receptor (CAR; GeneTex, Inc., Irvine, CA), bile salt export pump (BSEP), CYP7B1, CYP8B1, farnesoid X receptor (FXR), pregnane X receptor (PXR), small heterodimer partner (SHP), sulfotransferase (SULT)2A1, transforming growth factor (TGF)-β1 (Santa Cruz Biotechnology, Santa Cruz, CA), or multidrug resistance-associated protein 3 (MRP3; Sigma-Aldrich Japan). GAPDH (Santa Cruz Biotechnology) and TATA binding protein (TBP; Abcam plc) were used as loading controls for homogenates and nuclear fractions, respectively. Protein signals were detected using ECL Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).

All stocks were stored at −20 °C. scOHCs were from Avanti (Alabama, US) and stored as 10 mM stocks in nitrogen flushed ethanol: 24OHC (#700061), 25OHC (#700019), 26OHC (#700021). Epirubicin (Cayman, UK Cat:12091) was stored at 10 mM in nuclease free water and protected from light. GSK2033 (ToCris, Abindon, UK – #5694) at 20 mM diluted in ETOH. ABC inhibitors: MK-571 (Cambridge Bioscience – Cat: 10029–1mg-CAY) and KO143 (Sigma – Cat: K2144–1mg) were diluted in DMSO, while Verapamil (Insight Biotechnology – Cat: sc-3590) was diluted in NFW; all at 10 mM. TaqMan assays (Thermo Fisher, Paisley, #4331182): LXRα [Hs00172885_m1] LXRβ [Hs01027215_g1], Pgp [Hs00184500_m1], ABCA1 [Hs01059137_m1], HPRT1 [Hs02800695_m1]. Origine trisilencer complexes (Maryland, US): LXRα #SR322981, LXRβ #SR305039). Antibodies: Pgp (Santa Cruz Biotech, CA, US - #sc73354), CYP27A1 (Abcam, Cambridge, UK - #ab126785), CYP46A1 (Abcam, Cambridge, UK - #ab198889), CH25H (Bioss, MA, US - #bs6480R), LXRα (R&D System, Minneapolis, US - #PP-PPZ0412-00), LXRβ (Active Motif, Carslbad, US - #61177). Antibody validation is described in detail in Supplementary Information.