5z 7 oxozeaenol

Manufactured by Bio-Techne

Sourced in United Kingdom

(5Z)-7-Oxozeaenol is a lab equipment product offered by Bio-Techne. It is a small molecule that acts as a potent and selective inhibitor of TAK1 (Transforming growth factor-beta-activated kinase 1).

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Lab products found in correlation

Sb 505124, by Merck Group (2 mentions) Sb431542, by Bio-Techne (2 mentions) Mouse recombinant noggin, by ProSpec (2 mentions) Actinomcyin d, by Merck Group (2 mentions) Human recombinant bmp4, by Merck Group (2 mentions) Dorsomorphin, by Bio-Techne (2 mentions) Dimethyl sulfoxide (dmso), by Bio-Techne (1 mentions) Af 241 na, by R&D Systems (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) Melatonin, by Merck Group (1 mentions) Aspirin, by Bio-Techne (1 mentions) Pd153035, by Bio-Techne (1 mentions) Ag490, by Bio-Techne (1 mentions) P smad2 antibody, by Cell Signaling Technology (1 mentions) Arecoline, by Merck Group (1 mentions)

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TAK1 inhibitor (5Z)-7-Oxozeaenol (4 citations)

19 protocols using 5z 7 oxozeaenol

Urate-Primed Monocyte Cytokine Secretion

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For urate priming experiments, adherent monocytes were primed for 24 h in RPMI supplemented with 10% human pool serum with or without urate (Sigma, 69-93-2) and recombinant TGF-β1 (R&D Systems, Catalogue number 7754-BH-005). After 24 h, cells were restimulated with 10 ng/mL ultra-pure E. coli LPS (InVivogen, Catalogue number tlrl-pelps). Subsequently, cell-free supernatants were collected. Secretion of cytokines was measured in supernatants using ELISA kits for IL-1β, IL-6, IL-1Ra and TGF-β (R&D Systems, Catalogue number DY201, DY206, DY280 and DY240 respectively).
To inhibit TGF-β receptor signalling, three inhibitors were used. The ALK4/5/7-kinase inhibitor SB-505124 (Sigma) in a concentration of 5 μM with DMSO as solvent control, 5Z-7-oxozeaenol (100 nM) dissolved in DMSO (Tocris Bioscience) and a blocking antibody against TGF-β receptor II (AF-241-NA, R&D systems) with mouse IgG1 as the isotype control (10 μg/mL). Cells were pre-incubated with the inhibitor for 0.5 h before adding urate.

Klück V., Cabău G., Mies L., Bukkems F., van Emst L., Bakker R., van Caam A., Crişan T.O, & Joosten L.A. (2023). TGF-β is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells. Arthritis Research & Therapy, 25, 30.

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Molecular Mechanisms Regulating Cell Signaling

Cited 2 times

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Dulbecco’s modified Eagles’ medium (DMEM), penicillin/streptomycin, trypsin/EDTA, fetal bovine serum (FBS), KGM-SFM medium, and phosphate-buffered saline (PBS) were from Gibco (Life Technologies, USA). Arecoline, melatonin and MTT were from Sigma (Sigma-Aldrich Chemical Company, St. Louis, MO, USA). ELISA kits for MMP-9 were from PeproTech (PeproTech, Inc., Rocky Hill, NJ, USA). Catalase, SB431542, 5Z-7-Oxozeaenol, PD153035, AG490, U0126, LY294002, and aspirin were purchased from Tocris. Phospho-TAK1 (p-TAK1) antibody was from Abcam (ab79583). Antibodies for TGF-β1, and GAPDH were from Santa Cruz. P-Smad2 antibody was from Cell Signaling Technology. PBL extract and ANE were prepared as before [8 (link), 12 (link), 13 (link), 21 (link)]. HC was synthesized as previously [22 (link)].

Chang M.C., Pan Y.H., Wu H.L., Lu Y.J., Liao W.C., Yeh C.Y., Lee J.J, & Jeng J.H. (2019). Stimulation of MMP-9 of oral epithelial cells by areca nut extract is related to TGF-β/Smad2-dependent and –independent pathways and prevented by betel leaf extract, hydroxychavicol and melatonin. Aging (Albany NY), 11(23), 11624-11639.

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Detailed Compound Sourcing Protocol

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Unless otherwise stated, all compounds were obtained from Sigma-Aldrich. All other chemicals were of the highest commercial grade available. 5Z-7-oxozeaenol was acquired from Tocris Bioscience. All stock solutions were produced in non-pyrogenic saline (0.9% NaCl, Baxter, Milan, Italy).

Campolo M., Lanza M., Casili G., Paterniti I., Filippone A., Caffo M., Cardali S.M., Puliafito I., Colarossi C., Raciti G., Cuzzocrea S, & Esposito E. (2020). TAK1 Inhibitor Enhances the Therapeutic Treatment for Glioblastoma. Cancers, 13(1), 41.

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Immunohistochemical Analysis of Neuronal Signaling

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The antibodies and reagents used in this study were as follows: GFP (A6455; rabbit; Invitrogen; Chen et al., 2011 (link)); MAP2 (AB5622; rabbit; EMD Millipore; Chen et al., 2011 (link)); MAP2 (M4403; mouse; Sigma-Aldrich; Chen et al., 2011 (link)); SMI-312R (SMI-312R; mouse; Covance; Liu et al., 2013 (link)); HA (3F10; rat; Roche; Chen et al., 2017 (link)); phospho-P38 MAPK (9211; rabbit; Cell Signaling Technology); rabbit polyclonal P38 MAPK antibody (9212; rabbit; Cell Signaling Technology); phospho-ERK (4376; rabbit; Cell Signaling Technology); ERK (4695; rabbit; Cell Signaling Technology); phospho-JNK (9251; rabbit; Cell Signaling Technology); JNK (9252; rabbit; Cell Signaling Technology); phospho-TAK1 (9339; rabbit; Cell Signaling Technology); GAPDH (sc-25778; rabbit; Santa Cruz Biotechnology, Inc.; Chen et al., 2011 (link)); NeuN (MAB377; mouse; EMD Millipore; Wang et al., 2015 (link)); HRP-conjugated secondary antibodies (GE Healthcare); and Alexa Fluor 488– and Alexa Fluor 594–conjugated secondary antibodies (Invitrogen). Antibodies with validation profiles in Antibodypedia or 1DegreeBio are underlined. CL075, poly dT, poly(I:C) high molecular weight, and SB203580 were all purchased from InvivoGen. Takinib and NG 25 were purchased from Medchem Express, and (5Z)-7-oxozeaenol was purchased from Tocris.

Hung Y.F., Chen C.Y., Shih Y.C., Liu H.Y., Huang C.M, & Hsueh Y.P. (2018). Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs. The Journal of Cell Biology, 217(8), 2727-2742.

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Inhibition of TGF-β Signaling Pathways

Cited 1 time

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Before stimulation, cells were deprived of serum for 24 h and thereafter stimulated with 1 ng/ml recombinant human TGFβ1 (Biolegend, the Netherlands) for 2 or 24 h. In experiments where inhibitors were used, DMSO was used as vehicle control. To block TAK1 activity, we used (5Z)-7-Oxozeaenol [17 (link)] (Tocris Bioscience) in a concentration of 0.5 μM. To inhibit ALK5 kinase, we used SB-505124 [15 (link)] (Sigma Aldrich) in a concentration of 5 μM. For inhibition of ALK1 kinase, LDN-193189 [14 (link)] (Axon Medchem) was used in a concentration of 0.05 μM. This concentration of LDN-193189 is well above its reported half maximal inhibitory concentration (IC₅₀), 0.8 nM for ALK1, but far below its IC₅₀ for ALK5 of 350 nM [16 (link), 23 (link)]. Cells were pre-incubated with the inhibitors for 1 h prior to addition of TGF-β1. Either 2 or 24 h after addition TGF-β1, medium was removed and TRI-reagent was added for RNA isolation.

van Caam A., Madej W., Garcia de Vinuesa A., Goumans M.J., ten Dijke P., Blaney Davidson E, & van der Kraan P. (2017). TGFβ1-induced SMAD2/3 and SMAD1/5 phosphorylation are both ALK5-kinase-dependent in primary chondrocytes and mediated by TAK1 kinase activity. Arthritis Research & Therapy, 19, 112.

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Investigating Immune Cell Signaling Pathways

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The following inhibitors were used: cytochalasin D (Sigma‐Aldrich) 5 μg/mL; bafilomycin A1 (Sigma‐Aldrich) 10 nM; IKK inhibitor VII (Merck‐Millipore, Darmstadt, Germany) 5 μM; (5Z)‐7‐oxozeaenol (Tocris, Bristol, UK) 0.75 μM. The effect of inhibitors on THP1 cell viability is shown in Supplementary Fig. 4. The following TLR agonists were used at 1 μg/mL unless otherwise indicated: Pam3CSK4, HKLM, Poly(I:C), LPS E. coli K12, flagellin Salmonella Typhimurium, FSL‐1, Imiquimod, ssRNA40/Lyovec, and ODN2006 (all InvivoGen, San Diego, CA, USA). The following cytokines were used: M‐CSF (R&D Systems) 50 ng/mL; IFN‐γ (Miltenyi Biotec) 1000 U/mL; interferon‐α2a (Miltenyi Biotec) 1000 U/mL. The following blocking antibodies were used: anti‐MR1 (clone 26.5; TH) 10 μg/mL; anti‐IL‐12p40/70 (eBioscience, Hatfield, UK) 5 μg/mL; anti‐IL‐18 (MBL International, Woburn, MA, USA) 5 μg/mL.

Ussher J.E., van Wilgenburg B., Hannaway R.F., Ruustal K., Phalora P., Kurioka A., Hansen T.H., Willberg C.B., Phillips R.E, & Klenerman P. (2016). TLR signaling in human antigen‐presenting cells regulates MR1‐dependent activation of MAIT cells. European Journal of Immunology, 46(7), 1600-1614.

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Investigating Foxp3 Regulation by NLK

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The following antibodies were used: rat anti-Foxp3 clone PCH101 and mouse anti-Foxp3 clone eBio7979 (eBioscience), mouse anti-Flag (Sigma-Aldrich), mouse anti-hemaglutinin (HA) clone 12CAS, rabbit anti-NLK (H-100) and goat anti-Actin (I-19) (Santa Cruz Biotechnology), and anti-HSP90 was purchased from Professor Ineke Braakman (UMC Utrecht, Utrecht, the Netherlands).
The following reagents were used: Phos-tag™ Acrylamide (WAKO Chemicals GmbH), Okadaic acid, SB203580">SB203580, SP600125">SP600125, U-0126 and Rapamycin (Enzo Lifesciences), PKB inhibitor VIII (Calbiochem), 5Z-7-Oxozeaenol and BIO (Tocris Bioscience, Bristol, United Kingdom, λPPAse (New England BioLabs) and recombinant GST-NLK active protein (Sigma-Aldrich).

Fleskens V., Minutti C.M., Wu X., Wei P., Pals C.E., McCrae J., Hemmers S., Groenewold V., Vos H.J., Rudensky A., Pan F., Li H., Zaiss D.M, & Coffer P.J. (2019). Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells. Cell Reports, 26(13), 3600-3612.e6.

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THP-1 Cell Differentiation and Viability Assay

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9 × 10⁵ undifferentiated THP-1 cells per well were seeded in 6-well plates (TPP) in RPMI 1640 (Sigma-Aldrich) medium supplemented with 10% FCS (Sigma). Cells were treated with 0.25, 1 or 5 μM (5Z)-7-Oxozeaenol (5Z, Tocris) or DMSO for 1 h. PMA to a final concentration of 100 nM was added and cells present in the cell culture supernatant or adhered to the cell culture plastic were separately counted. Cells present in the supernatant were collected by centrifugation and resuspended in RPMI 1640 medium. An aliquot was mixed with trypan blue solution (LifeTechnologies) and cell viability and cell count were determined by using and automated cell counter (Countess, LifeTechnologies). Adhered cells were detached by incubation with a 5-fold concentrated ready-made trypsin solution (Biochrom) for 5 min. Trypsinization was quenched by addition of RPMI 1640 medium following by cell harvest and counting as described for cells in the supernatant.

Richter E., Ventz K., Harms M., Mostertz J, & Hochgräfe F. (2016). Induction of Macrophage Function in Human THP-1 Cells Is Associated with Rewiring of MAPK Signaling and Activation of MAP3K7 (TAK1) Protein Kinase. Frontiers in Cell and Developmental Biology, 4, 21.

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Investigating AMPK and MAPK Modulators

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Stock solutions of 5-amino-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR, Sigma-Aldrich, SE), 6,7-Dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662, Tocris, UK), metformin hydrochloride (Cayman Chemicals, UK), 6-[4-(2-Piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine (compound C, Sigma-Aldrich, SE), (5Z)-7-Oxozeaenol (Tocris Bioscience, UK) and Escherichia coli serotype O111∶B4 derived LPS (Sigma-Aldrich, SE) were prepared according to manufactures recommendations, aliquoted and stored at 20°C.
Various sets of experiments were performed. Incubations were for 4 days. Cultures were exposed to 1. LPS (0.2–20 µg/mL) with or without (5Z)-7-Oxozeaenol (10⁻⁶ M) or compound C (10⁻⁵ M), 2. (5Z)-7-Oxozeaenol (10⁻⁷–10⁻⁵ M), 3. compound C (3×10⁻⁷–3×10⁻⁵ M), 4. AICAR (10⁻⁴–3×10⁻³ M), A-769662 (10⁻⁵–3×10⁻⁴ M) or metformin (10⁻⁷–10⁻³ M), 5. AICAR (10⁻³ M), A-769662 (10⁻⁴ M) or metformin (10⁻⁴ M) together with compound C (10⁻⁵ M), 6. AICAR (10⁻³ M), metformin (10⁻⁴ M) or LPS (20 µg/mL) together with (5Z)-7-Oxozeaenol (10⁻⁶ M). Controls were always run in parallel.

Voss U, & Ekblad E. (2014). Lipopolysaccharide-Induced Loss of Cultured Rat Myenteric Neurons - Role of AMP-Activated Protein Kinase. PLoS ONE, 9(12), e114044.

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Salinomycin Effects on ARPE-19 Cells

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Human ARPE-19 cells (ATCC, Manassas VA) were grown in HEPES-buffered DMEM and Ham’s F12 (1:1) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% anti-anti (Life Technologies) at 37°C/5% CO₂. Cells (between passages 3 and 18) were plated at a density of 10,000 cells per well and grown in DMEM/F12 + 10% FBS for 24 hours, then starved in DMEM/F12 + 0.1% FBS for 16–18 hours before treatment to remove any response to TGFβ found in FBS. Salinomycin (Sigma, S4526) dissolved in DMSO was added to media with 0.1% FBS and then cells were assayed after 48 hours, unless otherwise indicated. SB-431542 (Sigma) and (5Z)-7-oxozeaenol (Tocris) were dissolved in DMSO and used at concentrations of 10uM and 1uM, respectively. Human primary RPE cells (Sciencell) were grown in EpiCM media supplemented with 2% FBS; starved cells were treated in EpiCM with no FBS added. Cells were plated and treated the same as ARPE-19 cells.

Heffer A.M., Proaño J., Roztocil E., Phipps R.P., Feldon S.E., Huxlin K.R., Sime P.J., Libby R.T., Woeller C.F, & Kuriyan A.E. (2019). The polyether ionophore salinomycin targets multiple cellular pathways to block proliferative vitreoretinopathy pathology. PLoS ONE, 14(9), e0222596.

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