Plasma from patients with normal lipid profiles and hyperlipidemia were supplied by Dr. John Kane and were collected under protocol # 10-00272 as approved by the Institutional Review Board at the University of California at San Francisco. Informed consent was obtained from all human subjects. Triglyceride rich lipoproteins (TRLs) were isolated as previously described25 (link). TRLs were quantified as previously reported26 (link). 2.5 nM LPL, final concentration, was incubated with isolated lipoproteins in a volume of 30 µL. Individual reactions were quenched at 0, 3, 6, and 9 minutes by the addition 10 µL Orlistat (Cayman Chemical Company, Ann Arbor, Michigan, USA) to a final concentration of 150 µM Orlistat in 40 µL. Released free fatty acids (FFA) were quantified using reagents previously described26 (link). Initial velocities were calculated by plotting FFA (µM) release against time from 0–9 minutes. Initial velocities were plotted as a function of substrate concentration and fit to the Michaelis–Menten equation using Kaleidagraph to calculate the kinetic parameters Vmax and Km. Each LPL sample was assayed at least five times.
Orlistat
Manufactured by Cayman Chemical
Sourced in United States
Orlistat is a lipase inhibitor used in the laboratory setting for research purposes. It functions by reducing the absorption of dietary fats.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions)
Ifn γ, by R&D Systems (3 mentions)
Human chylomicrons, by Abcam (2 mentions)
Am 1638, by Eli Lilly (2 mentions)
Gw1100, by Merck Group (2 mentions)
Recombinant mouse m csf, by R&D Systems (2 mentions)
7 aminoactinomycin d 7 aad, by BioLegend (2 mentions)
Chloroquine clq, by Merck Group (2 mentions)
Sulfo n succinimidyl oleate, by Merck Group (2 mentions)
Bafilomycin a1 baf, by Bio-Techne (2 mentions)
5 tetradecyloxy 2 furoic acid tofa, by Merck Group (2 mentions)
Etomoxir eto, by Merck Group (2 mentions)
Triacsin c, by Cayman Chemical (2 mentions)
Pik 3, by Cayman Chemical (2 mentions)
Vps34 in1, by Cayman Chemical (2 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Il 4 and il 13, by R&D Systems (1 mentions)
Lipopolysaccharides (lps), by Alexis Biochemicals (1 mentions)
Ova peptide, by InvivoGen (1 mentions)
Sorafenib, by Cayman Chemical (1 mentions)
20 protocols using orlistat
1
Lipoprotein Lipase Kinetics in Hyperlipidemia
2
Bone Marrow Derived Macrophage Differentiation and Stimulation
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Bone marrow cells were plated to a density of 5 x 106 cells per 90-mm bacterial Petri dish (Sterilin) in 10ml of DMEM/F-12 Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 with GlutaMAX supplement (Gibco) supplemented with 10% FBS, antibiotics, 20% L-cell conditioned medium, L-Glutamine (1%), HEPES (1%), Sodium Pyruvate (1%) and β-mercaptoethanol (1%). After 4 days of culture, 10ml of additional medium was added and cells were cultured for a further 3 days. Non-adherent cells were washed away and the adherent cells were collected in 5ml PBS with 5% FBS and 2.5mM EDTA. For experiments the cells were re-plated in medium with 1% FBS without L-cell supplement and were incubated overnight before stimulation. Cells were stimulated with IL-4 and IL-13 (20ng/ml) (R&D systems) or LPS (100ng/ml) (Alexis Biochemicals). In some experiments, BMDM were generated from three individual mice and co-cultured with a pool of naïve OTII CD4+CD44−Il4gfp–T cells during stimulation with OVA peptide (323–339) (Invivogen). In some of these co-culture experiments, BMDM were pre-treated for 6 hours with Orlistat (100μM; Cayman), prior to co-culture.
3
Sorafenib and Orlistat Solubilization
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Sorafenib (#10009644) and orlistat (#10005426) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Sorafenib and orlistat were dissolved in DMSO and absolute ethanol, respectively. The stock solutions of Sorafenib (40 mM) and orlistat (20 mM) were stored at −20 °C for the following experiments.
4
Investigating Anaplasma Infection Inhibition
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MβCD (Sigma) or orlistat (Cayman Chemical, Ann Arbor, MI) was added to the Anaplasma -infected cells at the indicated time postinfection and was retained in the growth medium throughout the incubation period or was removed at the time points as indicated in the figures and figure legends. Treatments at the concentrations used did not affect host cell viability and growth as assessed by trypan blue staining and light microscopy. For some experiments, host cell-free A. phagocytophilum purified from infected HL-60 was pretreated with the chemical in growth medium without fetal bovine serum (FBS) for 30 min at 37°C. The chemical was then removed by washing, and the bacteria were incubated with host cells. The degree of bacterial infection in host cells was assessed as described above.
5
Lipase Inhibition and FFA1 Modulation in Chylomicron Secretion
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All drugs and chemicals were purchased from Sigma-Aldrich (UK), unless otherwise stated. Drugs were made up as 1000× or 10,000× stock solutions, as per the manufacturer instructions. Human chylomicrons were obtained from BioVision (Milpitas, CA, USA), and ten individual batches were used during these studies. The broad-spectrum lipase inhibitor orlistat was manufactured by Cayman Chemical (Ann Arbor, MI, USA). The FFA1 agonist AM-1638 was kindly donated by Lilly (Indianapolis, IN, USA). The FFA1 antagonist GW1100 was manufactured by Merck Chemicals (Nottingham, UK).
The assay buffer used in secretion and imaging experiments contained (in mmol/l) 138 NaCl, 4.5 KCl, 4.2 NaHCO3, 1.2 NaH2PO4, 2.6 CaCl2, 1.2 MgCl2 and 10 HEPES (adjusted to pH 7.4 with NaOH).
The assay buffer used in secretion and imaging experiments contained (in mmol/l) 138 NaCl, 4.5 KCl, 4.2 NaHCO3, 1.2 NaH2PO4, 2.6 CaCl2, 1.2 MgCl2 and 10 HEPES (adjusted to pH 7.4 with NaOH).
6
Macrophage Polarization and Survival Assay
7
Comprehensive Metabolic Profiling Assay
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Sodium acetate (Sigma-Aldrich, S2889), C646 (Sigma-Aldrich, SML0002), orlistat (Cayman, 10005426), ACSS2 inhibitor (Millipore, 533756), mithramycin A (Cayman, 11434), pentamidine (Cayman, 20679), 14C2-acetic acid, (Perkin Elmer, NEC553050UC), 2-deoxyglucose (Sigma-Aldrich, D8375), 2,4-dinitrophenol (Sigma-Aldrich, D198501), rotenone (Sigma-Aldrich, cat. no. R8875), DAPI (Invitrogen, P36935), Turbofect transfection reagent (Thermo Fisher, R0532), DharmaFECT 1 transfection reagent (Horizon Discovery, T-2001), Nile red (Thermo Fisher, N1142), protein G magnetic beads (Thermo Fisher, 10004D), dispase II (Sigma-Aldrich, D4693), delipidated serum (Biowest, S181L), TRIzol (Thermo Fisher, 15596026), MG132 (EMD Millipore, 474790), PowerUP SYBR Green Mastermix (Applied Biosystems, A25742), MTT (Sigma-Aldrich, M2128), HA-magnetic beads (Thermo Fisher, 88836).
8
Macrophage Polarization and Survival Assay
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Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or lipopolysaccharide (LPS, 20 ng/ml; Sigma) plus IFN-γ (50 ng/ml; R&D Systems) in the presence or absence of 200 μM etomoxir (ETO; Sigma), 20 μM sulfo-n-succinimidyl oleate (SSO), 30 μM chloroquine (CLQ; Sigma), 0.1 μM bafilomycin A1 (Baf; Tocris Bioscience), 100 μM Orlistat (Cayman), 20 μM 5-(Tetradecyloxy)-2-furoic Acid (TOFA, Sigma), 5 or 50 μg/ml of LDL (Kalen Biomedical, LLC) and VLDL (Kalen Biomedical, LLC) for 24 h. Macrophages were then harvested and analyzed by flow cytometry for expression of markers of M1 or M2 activation. In some experiments the survival of macrophages was monitored following M2 or M1 activation. For these experiments, bone marrow-derived macrophages (0.5 × 106 cells per well of a 48-well plate) were cultured in complete medium with mouse M-CSF (20 ng/ml) and stimulated with IL-4 or LPS plus IFN-γ. Cell viability was monitored by flow cytometric analysis of 7-amino-actinomycin D (7-AAD; BioLegend) staining of F4/80+ cells over time. Culture medium and stimulation signals were replenished every 1 or 2 days.
9
GENS Extract Modulates Adipogenesis Factors
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GENS extract was obtained from NEWTREE Inc. (Seongnam, Kyeonggi, Korea). The composition of GENS extract is described in Table 1 . The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): CCAAT/enhancer binding protein alpha (C/EBPα, SC-61), diacylglycerol O-acyltransferase-1 (DGAT-1, SC-32861), fatty acid synthase (FAS, SC-20140), peroxisome proliferator activated receptor gamma (PPARγ, SC-7273), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, SC-25778), sterol regulatory element binding protein-1 (SREBP-1, SC-366), and uncoupling proteins-1 (UCP-1, SC-6529). PR domain-containing16 (PRDM16, ab106410) were obtained from Abcam (Cambridge, MA, USA). Phospho-adenosine monophosphate-activated protein kinase (p-AMPK, CS-2603s), AMPK (CS-2603s), phospho-acetyl-CoA carboxylase (p-ACC, CS-3661s), and ACC (CS-3662s) were purchased from Cell Signaling Technology (Bedford, MA, USA). Orlistat and metformin were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Glucose and methylcellulose were purchased from Sigma (Sigma, St. Louis, MO, USA). All chemicals and reagents used were of analytical and obtained from commercial sources.
10
Anticancer Compounds in Colorectal Cancer Cells
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DMEM (Dulbecco’s Modified Eagle’s Medium) was obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT). Cell-culture plates were purchased from Denville Scientific (Charlotte, NC). Celecoxib and Orlistat were obtained from Cayman Chemical (Ann Arbor, MI). Cell Titer 96 Aqueous One Solution Cell Proliferation assay was obtained from Promega (Madison, WI). The enhanced chemiluminescence reagents (ECL) kit and Pierce BCA protein assay were obtained from ThermoScientific (Rockford, IL). BD Pharmigen FITC Annexin V Apoptosis Detection Kit I was obtained from BD Biosciences (San Jose, CA). Control, MUC2, and CHOP shRNA lentiviral particles were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). For western blotting Beta-Actin (A1978) was obtained from Sigma Aldrich, St. Louis, MO; PARP-1 (CST 46D11), GRP78 (CST C50B12), ATF4 (CST D4B8), PUMA (CST D30C10), NOXA (CST D8L7U), CHOP (CSTL63F7), and Cleaved Caspase 3(CST Asp175) were obtained from Cell Signaling Technologies (Danvers, MA). SYTOX Orange for nucleic acid labeling was obtained from Life Technologies (Grand Island, NY). Anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson Immunology Research (West Grove, PA).
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