The generation and analysis of Hupki P72 and R72 mice, and backcrossing in a C57Bl/6J background for over ten generations, was described previously (Frank et al., 2011 (link)). The experiments with mice complied with all federal and institutional guidelines as per IACUC protocols. Mice were housed in plastic cages with ad libitum diet and maintained at 22°C with a 12-hour dark/12- hour light cycle. Wild-type C57Bl/6 mice were purchased from the Jackson laboratory (Bar Harbor, ME). Normal chow diet was obtained from LabDiet (Pico-Vac Diet 20, 3.75kcal/gm, 21% fat calories). The high-fat diet was customized and purchased from Harlan laboratories (TD. 130886, 4.4 kcal/g, 49% fat calories). Mouse weight and food consumption were monitored weekly throughout the duration of the experiments. Body composition (lean and fat mass) was measured with nuclear magnetic resonance spectroscopy (Echo MRI 3-in-1 analyzer, Houston, TX). For rescue experiments with inhibitors, prior to 7-day exposure to a high fat diet, 5-week old mice were weighed and then administered with TNF-α inhibitor C87 (10mg/kg; EMD Millipore, 530796) or NPC1L1 inhibitor Ezetimibe (10mg/kg; Cayman Chemical, 16331) via i.p. injection and oral gavage, respectively. Treatments were performed daily, and mice were weighed and euthanized for liver harvesting by the end of 7-day period.
Ezetimibe
Manufactured by Cayman Chemical
Sourced in United States
Ezetimibe is a laboratory product manufactured by Cayman Chemical. It is a selective inhibitor of intestinal cholesterol absorption.
Automatically generated - may contain errors
Lab products found in correlation
Wild type c57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Recombinant mouse ifn β, by R&D Systems (1 mentions)
Antibody to ch25h, by Bioss Antibodies (1 mentions)
Facscanto 2, by BD (1 mentions)
Progesterone, by Merck Group (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Anti f4 80 antibody, by Abcam (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Dulbecco s modified eagle s medium dmem, by GE Healthcare (1 mentions)
Rpmi 1640 culture medium, by GE Healthcare (1 mentions)
Triglyceride assay kit, by BioAssay Systems (1 mentions)
Mcc950 pz0280, by Merck Group (1 mentions)
6 protocols using ezetimibe
1
Generation and Analysis of Hupki P72 and R72 Mice
2
Transgenic Tomatoes Expressing 6F Peptide
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Transgenic tomatoes expressing the 6F peptide (Tg6F) were grown and processed as described (10 (link)). Ezetimibe was from Cayman Chemical Co. (Ann Arbor, MI, catalog no. 16331). Recombinant mouse IFN-β was from R&D Systems (Minneapolis, MN, catalog no. 8234-MB). Antibody to IFN-β was from Thermo Scientific (Waltham, MA, catalog no. PA5-20390). Antibody to CH25H was from Bioss Antibodies (Woburn, MA, catalog no. bs-6480R). Unless otherwise stated, all other materials were from sources previously described (14 (link)).
3
Cholesterol Modulation through Compounds
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4
Dose-dependent Ezetimibe Cytotoxicity Analysis
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We performed preliminary dose-dependent tests with Ezetimibe (Cayman Chemical Company, Ann Arbor, MI, USA) in order to identify the appropriate experimental conditions. THP-1 cells and human cardiomyocytes were incubated overnight with increasing amounts of Ezetimibe (from 5 to 50 μM). Cellular viability was evaluated with the Annexin V staining assay (BD Bioscience, Franklin Lakes, NJ, USA) as previously reported [19 (link)] and analyzed by flow cytometry on FACSCantoII (Becton Dickinson). The number of each type of cell was expressed as a percentage of the number of total stained cells.
5
Inhibitor treatments of E. bovis-infected BUVEC
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For inhibitor treatments, E. bovis-infected and non-infected BUVEC (n = 3 or 4) were treated with pharmacological blockers from 10 d p.i. onwards. Hence, BUVEC were treated with ezetimibe (20 µM; Cayman Chemical, Ann Arbor, MI, USA), ezetimibe-glucuronide (30 µM; Santa Cruz Biotechnology, Inc., Heidelberg, Germany), polycytidylic acid potassium salt (poly-C; 100 µg/ml; Sigma-Aldrich, P4903), polyinosinic acid (poly-I; 100 µg/ml; Sigma-Aldrich, P4154), and sucrose (100 mM; Sigma-Aldrich, S7903) serving as inhibitors of sterol uptake. Additionally, progesterone (25–75 µM; Sigma-Aldrich, P0130) and U18666A (7.5 µM; Cayman Chemical Company, 10009085) were used, which are both known to block intracellular sterol transport and release from endosomes. As solvent control, either water, ethanol, or dimethyl sulfoxide was used.
6
Inflammatory Response and Lipid Metabolism
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Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 culture medium and penicillin–streptomycin were purchased from GE Healthcare Life Sciences (Marlborough, MA, USA). IL-1β ELISA kit (Human 88-7261-22) and TRIzol reagent (15596018) was purchased from Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide (LPS; L6529), palmitate (P9767) and MCC950 (PZ0280) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pioglitazone was purchased from Takeda Pharmaceutical (AD-4833, Kanagawa, Japan). Ezetimibe was obtained from Cayman Chemical (16331, Ann Arbor, MI, USA). The triglyceride assay kit was purchased from Bioassay systems (ETGA-200, Hayward, CA, USA). Anti-F4/80 antibody was obtained from Abcam (ab6640, Cambridge, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Georgiachem (MT 1036, Norcross, GA, USA).
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