Pf 06260933

Immortalized human hepatocytes (IHHs; a gift from B. Staels, the Pasteur Institute of Lille, University of Lille Nord de France, Lille, France) (32 (link)) were maintained in Complete William's E Medium (GlutaMAX supplemented; Gibco, Paisley, UK) supplemented with dexamethasone (50 nmol/l; Sigma-Aldrich, St. Louis, MO), human insulin (20 U/l; Actrapid Penfill; Novo Nordisk, Bagsværd, Denmark), 10% (vol/vol) FBS, and 1% (vol/vol) penicillin/streptomycin (Gibco). Cells were demonstrated to be free of mycoplasma infection by the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).
IHHs were transfected with MAP4K4 small interfering (si)RNA (Hs.701013; Ambion, Austin, TX) or scrambled siRNA (SIC001; Sigma-Aldrich) using Lipofectamine RNAiMax (Thermo Fisher Scientific). Cells were also transfected with human MYC-tagged MAP4K4 expression plasmid (EX-A2245-M43; GeneCopoeia, Labomics, Nivelles, Belgium) or an empty control plasmid (EX-NEG-M43; GeneCopoeia) using Lipofectamine 2000 (Thermo Fisher Scientific). At 24 h posttransfections, the culture medium was replaced with fresh medium, with or without supplementation of 50 μmol/l oleic acid (Sigma-Aldrich), for subsequent 48 h incubation. In one experiment, MAP4K4-overexpressing cells were treated with a small-molecule MAP4K4 inhibitor PF-06260933 (Sigma-Aldrich (33 (link))).

LnCaP and 22rv1 PCa cell lines obtained from ATCC (Manassas, VA, USA) were kindly donated by Dr. Guillermo Velasco. PC3 and DU145 cell lines were obtained from ATCC (Manassas, VA, USA). LnCaP, 22rv1 and DU145 were cultured in RPMI 10% FBS. PC3 were cultured in RPMI/F12 10% FBS. HEK-293 T cells cultured in 10% FBS DMEM medium were used for virus amplification. To trigger HGK signaling cells were treated with TNF-α (20 ng/mL; R&D, Minneapolis, MN, USA). The chemical inhibitor used to inhibit HGK was PF 06,260,933 (1–10 µM; Sigma, PZ0272).

At 6–8 weeks of age, male flox/flox and flox/flox/cre+ littermates were injected with 1 mg tamoxifen per day in corn oil (Sigma) for 5 days. At 5–6 weeks of age (KD mice) or 2 weeks after tamoxifen injection (flox mice), the mice were fed chow or WD (0.2% cholesterol, TD 88137, Harlan Laboratories) for 16 weeks. Compound PF-06260933 Sigma Aldrich (catalog # PZ0272) (10 mg kg⁻¹, dissolved in dH₂O) was orally administered to 8–10-week-old male ApoE^−/− mice twice daily for 6 weeks. Ldlr^−/− male mice (B6.129S7-Ldlr^tm1Her/J, Jackson Laboratories, 8–10 weeks old) were placed on HFD (1.25% cholesterol, TD96121, Harlan Laboratories) for 10 weeks before drug administration. Compound PF-06260933 was administered to male 8–10-week-old Ldlr^−/− mice as above for 10 weeks. Oral administration of water was used as vehicle control in all studies. Mice were euthanized by CO₂ inhalation followed by bilateral pneumothorax. No statistical methods were used to predict sample size, no randomization was performed and the investigations were not blinded during the knockout animal analyses, but were blinded during the drug treatment analyses.