Racemic ±14-hydroxy-4Z,7Z,10Z,12E,16Z,19Z-DHAs (±14-HDHAs or 14S/R-HDHAs) and 5-lipoxygenase (5-LOX, human recombinant or potato) were supplied by Cayman (Ann Arbor, MI, United States). NaBH4, interleukin (IL)-1β, TNF-α, and Escherichia coli lipopolysaccharide (LPS) were supplied by Sigma-Aldrich (St. Louis, MO, United States).
Interleukin il 1β
Manufactured by Merck Group
Sourced in United States
Interleukin (IL)-1β is a cytokine that plays a key role in the inflammatory response. It is a product of activated macrophages and is involved in the regulation of immune responses, inflammation, and hematopoiesis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Escherichia coli lipopolysaccharide lps, by Merck Group (1 mentions)
Tnf α, by Merck Group (1 mentions)
Nabh4, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Merck Group (1 mentions)
Chon 001, by Merck Group (1 mentions)
Bay 11 7085, by MedChemExpress (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Chon 001, by American Type Culture Collection (1 mentions)
β actin antibody, by Fortis Life Sciences (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt dexamethasone, by Merck Group (1 mentions)
Lamin b1 antibody, by Bioworld Technology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
5 protocols using interleukin il 1β
1
Synthesis and Evaluation of 14-HDHAA Compounds
2
Transient Knockdown of CUGBP1 in A549 Cells
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A549 cells were seeded at a density of 5 × 105 cells/well in a 6-well plate and treated with 20 pmol siRNA oligonucleotides the next day using Lipofectamine 2000® (Invitrogen) according to manufacturer’s instructions. A previously published anti-CUGBP1 siRNA (5′-GCUGUUUAUUGGUAUGAUU-3′) was used for transient knockdown (Δ) of CUGBP1 (Vlasova-St Louis and Bohjanen, 2011 (link)). As control, a scramble siRNA with an unspecific sequence (5′-UCUCUCACAACGGGCAUUU-3′) was used. 24 h after transfection, A549 cells were incubated with 5 ng/ml of Interleukin (IL)-1β (Sigma-Aldrich) for further 24 h. The efficiency of the knockdown was determined by Western blot analysis as described in Saul et al. (2019a) (link).
3
Chondrocyte Model of Osteoarthritis
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The human chondrocyte cell line CHON-001 and 293T cells were obtained from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 2 mM glutamine (Sigma-Aldrich; Merck KGaA) andplaced at 37˚C in a humidified incubator containing 5% CO2. CHON-001 cells were treated with 10 ng/ml interleukin (IL)-1β (Sigma-Aldrich; Merck KGaA) at room temperaturefor 24 h to generate an in vitro model of OA (16 (link)). The nuclear factor κB (NF-κB) inhibitor BAY 11-7085 (10 µM) was purchased from MedChemExpress.
4
Anti-inflammatory Compound K Effects
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2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride (AMT) was purchased from Tocris Cookson Ltd. (Avonmouth, Bristol, UK). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dexamethasone, interleukin (IL)-1β, and lipopolysaccharide (LPS, Escherichia coli 0127:B8) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Compound K-enriched fraction (5%) was obtained from Fleton Natural Products Co. (Chengdu, China). The protein assay kit was purchased from Bio-Rad Lab (Hercules, CA, USA). All antibodies relating to MAPK and nuclear transcription factor-κB (NF-κB) signaling were purchased from Cell Signaling Technologies (Dancers, MA, USA). β-actin antibody was obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA). Lamin B1 antibody was purchased from Bioworld technology (Minneapolis, MN, USA).
5
Culturing Synovial Fibroblasts and Stimulation
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The human embryonal kidney cell line (HEK 293) (DMSZ, ACC 035) and HeLa (DMSZ, ACC57) cells were cultured in Dulbecco's modified Eagle medium (DMEM, Life Technologies, ThermoFisher Scientific, Waltham, USA) with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Life Technologies, ThermoFisher Scientific, Waltham, USA), 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mM sodium pyruvate (PAA the Cell Culture Company, Cölbe, GER). Synovial fibroblasts (SFs) of RA patients were cultured as described previously (16 (link)). This study was approved by the Institutional Ethical Committee (Solna, Stockholm, Sweden ethical number 2009/1262-31/3) and is in compliance with all ethical standards and patients' consent according to the Declaration of Helsinki. Cell culture was carried out in a humidified atmosphere of 5% CO2 at 37°C. SFs were seeded at a density of 5 × 104 cells/well in 24-well plates and cultured in DMEM-medium supplemented with EV-free FBS (Gibco, Life Technologies, ThermoFisher Scientific, Waltham, USA), 100 U/ml penicillin 100 μg/ml streptomycin and 1 mM sodium pyruvate for 24 h before they were stimulated with 10 ng/ml ml interleukin (IL)-1β (Sigma-Aldrich, Darmstadt, GER) or tumor necrosis factor α (TNF α) (Immuno Tools, Friesoythe, GER) for further 24 h.
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