9 cis retinoic acid

Reagents were purchased from the following sources. Antibodies used are as follows: SREBP1 (2A4, Santa Cruz Biotechnology), SREBP2 (30682, Abcam), LXRα (PP-PPZ0412-00, R&D systems), LXRβ (K8917, R&D systems), phospho-T308 AKT (C31E5E), AKT (11E7) (Cell Signaling Technology).
Drugs used are as follows: Cycloheximide (Cell Signaling Technology) was used at 10 μg/ml. 9-cis-retinoic acid (Sigma) was used at 50 μM. The LXR antagonist GSK-2033 (Axon Medchem) was used at 500 nM. The LXR agonist GW3965 (Fisher Scientific) was used at 500 nM. PI3K inhibitor, LY294002 (Cell Signaling Technology), was used at 10 μM. Rapamycin was used at 100 nM and received as a gift from David Sabatini. Methyl-beta-cyclodextrin was purchased from Sigma. All sterols except for custom-synthesized ent-4β-HC (see below) were purchased from Steraloids. C13 glucose was purchased from Cambridge Isotope Laboratories.

Human fibronectin was purchased from BD Biosciences (Bedford, MA). The purified Human fibronectin alpha-chymotryptic fragment, FN-120, and the Fibronectin 40 kDa α Chymotryptic Fragment (Heparin-binding region), FN-40, was purchased from Millipore (Temecula, CA). Purified human vitronectin was purchased from R&D systems (Minneapolis, MN). The function blocking antibodies SAM-1 (anti-integrin α5), B-D15 (anti-integrin β1), F11 (anti-integrin β3), MOPC-21 (isotype control), and the secondary goat anti-human IgG FITC conjugated antibody were purchased from Abcam (Cambridge, MA). Anti-integrin αvβ3 and αvβ5 FITC conjugated antibodies were purchase from Millipore. The anti α8 integrin and the secondary rat anti-mouse IgG_2B FITC conjugated antibody was purchased from R&D systems. 9-cis-retinoic acid, 13-cis-retinoic acid, troglitazone, and p-nitro-phenyl phosphate were purchased from Sigma (St. Louis, MO). 4-oxo-retinoic acid was kindly provided by Hoffman-La Roche (Nutley, NJ). Because of retinoid photosensitivity, all experiments were performed under dim light. Samples and reference compounds were stored at −20°C or +4°C. Retinoids were dissolved at the desired concentration in ethanol. Other reagents used in the extraction process, analysis, or standard preparations were Optima grade hexane, methanol, HPLC grade water, and Tracemetal grade acetic acid.

T0901317, 9-cis-retinoic acid, phorbol 12-myristate 13-acetate (PMA), GLP-1 (7–37), and exendin-(9–39) were purchased from Sigma (St Louis, MO, USA). Endotoxin, fatty-acid-free bovine serum albumin (BSA), and the PKA inhibitor 14–22 amide were purchased from Calbiochem (Merck KGaA, Darmstadt, Germany). Sitagliptin phosphate and vildagliptin were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Drugs were selected based on literature mining of CYP2W1 induction [14 (link)], NCBI GEO database, and activity reported in some artificial recombinant models [18 –20 ]. We have also included modulators of the known transcriptional factors, such as AhR, CAR, PXR, PPARγ, RXR, RAR (S2 Table).
Linoleic acid (LA), conjugated (9Z,11E)-Linoleic acid (9Z11E-LA), conjugated (10E,12Z)-Linoleic acid (10E12Z-LA), citco, rifampicine, 9-cis-Retinoic acid (RA), all-trans-retinoic acid, ciglitazone, 5-Aza-2'-deoxycytidine (DAC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were purchased from Sigma-Aldrich (Stockholm, Sweden). Imatinib was obtained from Toronto Research Chemicals (North York, Canada) and GW610 from Cayman Chemical Comp (Ann Arbor, MI, USA).
HCC2998 cells were treated for 48 h with the vehicle (0.1% DMSO or 0.1% ethanol according to the drugs solubility, see S2 Table) or the indicated drugs. The culture medium, including vehicle and investigated drugs was exchanged every 24 h. After cell harvesting, the CYP2W1 mRNA levels were analyzed as described above. All experiments were performed at least twice in at least two replicates per experiment.