7β hydroxycholesterol

Human THP-1 monocytes (ATCC, VA, USA) were maintained in RPMI-1640 medium with glutamate supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO, UK). Cells were cultured in 5% CO₂ humidified atmosphere at 37°C and divided twice a week. For all experiments cells were seeded at an approximate density of 5 x 10⁵/mL. Macrophage differentiation was induced by incubation with 300 nM phorbol myristate acetate (PMA; Sigma-Aldrich, MO, USA) for 24 h. Culture medium was then replaced and cells cultured for an additional 48 h. Experiments were performed with a mixed treatment of 7β-hydroxycholesterol and 7-ketocholesterol (Sigma-Aldrich) at a ratio of 1:1.8 (28 μM; referred to as 2mix) for 24 hr, previously tested within our group to induce an apoptotic state [5 (link), 16 (link)]. Control experiments were performed using untreated, cholesterol, ethanol, and 7-ketocholestrol individually. The concentrations of oxysterols used in the experiments are substantially lower than those reported to exist in human atherosclerotic plaque [3 (link), 17 (link)], although greater than normal healthy physiological conditions, and provide a viable cell model to analyse macrophage dysfunction during atherosclerosis [4 (link)].

The contents of cholesterol and oxysterols were determined according to the slightly improved method of Czauderna et al. [32 (link)]. The improvements consisted of silylation with a BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide, 99%; Sigma Aldrich, St. Louis, MO, USA) procedure. Derivatized analytes were separated on a GC-TOFMS Pegasus^® BT (LECO Corporation, St. Joseph, MI, USA) chromatograph equipped with a capillary column (30 m × 0.25 mm × 0.25 μm film thickness, Rxi^®-17SilMS, Restek, Bellefonte, PA, USA). Identification was made on the basis of mass spectra and by a comparison of retention times of analytes with the following standards: cholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, cholesterol 5α,6α-epoxide, 7-ketocholesterol; Sigma, USA). For recoveries, 5α-cholestane (Sigma, St. Louis, MO, USA) as an IS was used.

Dulbecco's Modified Eagle Medium (DMEM) chemicals, monodansylcadaverine (MDC) and 7β-hydroxycholesterol were obtained from Sigma Aldrich Chemical (St. Louis, MO) as were all other reagents, unless specifically stated elsewhere. Fetal bovine serum (FBS) and penicillin-streptomycin were provided by Lonza (Basel, Switzerland). α-Asarone was purchased from Cayman chemical company (Ann Arbor, MI). Antibodies of eIF2α, phospho-eIF2α, Vps34, Atg5, Atg12-Atg5, Atg16L1, SQSTM1, NBR1, Atg7, Atg3, CHOP were obtained from Cell Signaling Technology (Danvers, MA). GADD34, beclin-1 antibodies were purchased from Abcam (Cambridge, UK). p150 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX). LC3 antibody was purchased from MBL international corporation (Woburn, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and rabbit anti-mouse IgG were supplied from Jackson ImmunoResearch Laboratory (West Grove, PA).

Olein, stigmasterol, β-sitosterol, campesterol, brassicasterol, cholesterol, 7α-Hydroxycholesterol, 7β-Hydroxycholesterol, 7-Ketocholesterol, 5α,6α-Epoxy-cholesterol, 5β,6β-Epoxycholesterol, cholestanetriol, 19-Hydroxycholesterol, and 5α-cholestane were purchased from Sigma–Aldrich. The derivatization reagents N-methyl-N-(trimethylsilyl) heptafluorobutyramide and 1-methyl imidazole were ordered from Sigma–Aldrich. Acetone, diethyl ether, dichloromethane, hexane, and methanol were obtained from Merck & Co, Inc. FeSO4·7H₂O, CuSO₄·5H₂O, ZnSO₄, MgSO₄, Na₂SO₄, and MnSO₄ were analytically pure.

All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA); GenMute^™ siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA). C57BL/6J mice were obtained from the Jackson Laboratory (Bar harbor, ME, USA) and cared for under housing and animal protocols approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.