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Uo126

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UO126 is a highly selective inhibitor of the mitogen-activated protein kinase kinase (MEK1/2) enzymes. It acts by blocking the phosphorylation and activation of ERK1/2 proteins, which are important components of the MAPK signaling pathway.

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Lab products found in correlation

Ly294002, by Cell Signaling Technology (4 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Fluorescent leptin, by PerkinElmer (2 mentions) Egf tritc, by Thermo Fisher Scientific (2 mentions) Fluorescent leptin, by Cell Signaling Technology (2 mentions) Nocodazole, by Merck Group (1 mentions) Collagen 3, by Abcam (1 mentions) Gapdh, by Abcam (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Human recombinant tgf β1, by Santa Cruz Biotechnology (1 mentions) Antibodies against α sma, by Abcam (1 mentions) Sb431542, by Cell Signaling Technology (1 mentions) Sp600125, by Cell Signaling Technology (1 mentions) Phospho c jun, by Cell Signaling Technology (1 mentions) Mk 2206, by Selleck Chemicals (1 mentions) Sb203580, by Cell Signaling Technology (1 mentions) Ve cadherin, by Abcam (1 mentions) Azd6244, by Selleck Chemicals (1 mentions) Refametinib, by Chemietek (1 mentions)

25 protocols using uo126

1

Nocodazole, PD0325901, and U0126 Treatments

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Nocodazole (Sigma M1404) was administered in 24 or 48 hr increments at 50 ng/mL. PD0325901 (EMD Millipore/Calbiochem 4449685 MG) and UO126 (Cell Signaling Technologies 9903S) were administered at 10 µM and 25 µM, respectively. Drugs were diluted in planaria water containing 0.05% DMSO. Animals were rinsed three times after treatment and either fixed immediately, or transferred to a new dish and rinsed daily until further experimentation.
Bohr T.E., Shiroor D.A, & Adler C.E. (2021). Planarian stem cells sense the identity of the missing pharynx to launch its targeted regeneration. eLife, 10, e68830.
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2

TGF-β1 Signaling Pathway Investigation

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Human recombinant TGF‐β1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against α‐SMA, VE‐cadherin, collagen I, collagen III, CD31, vWF and GAPDH were purchased from Abcam (Cambridge, MA, USA). Antibodies against phospho‐Smad 2 (Ser465/467), phosphor‐Smad 3 (Ser423/425), phospho‐Akt (Ser473), phospho‐mTOR (Ser2448), phospho‐p70S6K (Thr389), phospho‐Erk 1/2 (Thr202/Tyr204), phospho‐p38 MAPK (Thr180/Tyr182), phospho‐c‐Jun (Ser73), Smad 2, Smad 3, Akt, mTOR, p70S6K, Erk 1/2, p38 MAPK and c‐Jun, and selective inhibitors including SB431542, SB203580, UO126 and SP600125 were purchased from Cell Signaling Technology (Beverly, MA, USA). MK2206 was purchased from Selleckchem (Houston, TX, USA). Penicillin‐streptomycin and Roswell Park Memorial Institute (RPMI)‐1640 medium were purchased from Invitrogen (Life Technologies, Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA).
Wang Z., Han Z., Tao J., Wang J., Liu X., Zhou W., Xu Z., Zhao C., Wang Z., Tan R, & Gu M. (2017). Role of endothelial‐to‐mesenchymal transition induced by TGF‐β1 in transplant kidney interstitial fibrosis. Journal of Cellular and Molecular Medicine, 21(10), 2359-2369.
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3

Inhibition of MEK and PI3K Signaling

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The MEK inhibitor, UO126 (Cell Signaling Technology, Danvers, USA) and selumetinib AZD6244 (Selleck Chemicals) and the PI3K inhibitor, LY294002 (Euromedex, Mundolsheim, France) were used with the indicated concentration.
Buffet C., Hecale-Perlemoine K., Bricaire L., Dumont F., Baudry C., Tissier F., Bertherat J., Cochand-Priollet B., Raffin-Sanson M.L., Cormier F, & Groussin L. (2017). DUSP5 and DUSP6, two ERK specific phosphatases, are markers of a higher MAPK signaling activation in BRAF mutated thyroid cancers. PLoS ONE, 12(9), e0184861.
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4

Combinatorial Drug Cytotoxicity Assay

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Inhibitors were purchased from SelleckChem (H89.2HCl, BI‐D1870, LJH685, Trametinib) or Cell Signaling Technologies (UO126, LY294002, BYL‐719) and diluted in DMSO. Refametinib (Chemietek) was diluted in water.
For combinatorial treatments, cells were treated with the indicated inhibitors for 30 min before addition of cisplatin (Sigma) or carboplatin (SelleckChem). Cytotoxicity was assessed after 72 h using CellTiter‐Glo Luminescent Cell Viability Assay (Promega) for 5–20 min before luminescence detection.
Moyano‐Galceran L., Pietilä E.A., Turunen S.P., Corvigno S., Hjerpe E., Bulanova D., Joneborg U., Alkasalias T., Miki Y., Yashiro M., Chernenko A., Jukonen J., Singh M., Dahlstrand H., Carlson J.W, & Lehti K. (2020). Adaptive RSK‐EphA2‐GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer. EMBO Molecular Medicine, 12(4), e11177.
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5

Cardiac Stem Cell Isolation and Differentiation

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CSCs were isolated from adult transgenic and wt hearts by enzymatic methods.35 (link), 40 (link) For CD45−c-Kit+ cell purification, myocyte-depleted small cardiac cells were incubated with microbeads conjugated with anti-mouse CD45 antibody (Miltenyi Biotec S.r.l., Calderara di Reno (BO), Italy) and removed from the preparation by magnetic-activated cell sorting (Miltenyi). From the CD45− fraction, the c-Kit+ (CD45−) cardiac cells were enriched through incubation with a microbeads-conjugated mouse monoclonal antibody against c-Kit (Miltenyi) and separated by magnetic-activated cell sorting.
Freshly isolated CD45−c-Kit+ cardiac cells were cultured on gelatin-coated dishes in CSC growth medium35 (link), 40 (link) before clonogenic, spherogenic and BrdU assays.
Cardiosphere myogenic differentiation was performed as previously described.35 (link)Endothelial differentiation was obtained by culturing the CSC for 3–10 days in MEM Alpha (Life Technologies), 10% ESQ-FBS (Life Technologies), 1 μM dexamethasone, 50 μM/ml ascorbic acid, 10 mM β-glycerophosphate (all from Sigma-Aldrich, Milano, Italy) and 10 ng/ml VEGF (PeproTech EC Ltd., London, UK). LY294002 (10 μM, Calbiochem, San Diego, CA, USA) and UO126 (10 μM, Cell Signalling Technology, Danvers, MA, USA) were added to the culture for AKT and ERK1/2 inhibition.
Di Siena S., Gimmelli R., Nori S.L., Barbagallo F., Campolo F., Dolci S., Rossi P., Venneri M.A., Giannetta E., Gianfrilli D., Feigenbaum L., Lenzi A., Naro F., Cianflone E., Mancuso T., Torella D., Isidori A.M, & Pellegrini M. (2016). Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury. Cell Death & Disease, 7(7), e2317-.
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6

Modulation of Neutrophil Signaling Pathways

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In some studies, neutrophils were preincubated for 30 min at 37°C with various inhibitors in assay medium before running the assay. For analysis of signaling pathways, 10 µM UO126 (ERK inihibitor, Cell Signaling Technology, USA), 10 µM VIII (Akt inhibitor, Calbiochem, Germany), or 10 µM SB 203580 (p38 MAPK inhibitor, Calbiochem) were used. To inhibit the Na+/H+ exchanger (NHE), neutrophils were preincubated with 10 µM of the NHE-inhibitor DMA [5-(N,N-dimethyl)amiloride hypochloride, Sigma]. The H+ATPase (V-ATPase) was inhibited by adding 100 nM Bafilomycin A1 (InvivoGen, USA). The glycolysis pathway was inhibited by use of 10 mM of the hexokinase-inhibitor 2-deoxyglucose (2DG, Sigma) or 100 µM of the glyceraldehyse-3-phosphate-dehydrogenase inhibitor sodium idoacetate (SIA, Sigma). Neutrophils exposed to the appropriate solvent [dimethyl sulfoxide (DMSO)] concentrations and untreated neutrophils served as controls. All inhibitors were freshly prepared for each experiment. The used inhibitors and the final DMSO concentration (0.1% v/v) had no toxic effects on neutrophils within 7 h, as analyzed by the Annexin V-/PI staining (data not shown).
Behnen M., Möller S., Brozek A., Klinger M, & Laskay T. (2017). Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps. Frontiers in Immunology, 8, 184.
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7

Leptin Signaling Pathway Inhibition

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Recombinant human leptin and AG490 were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). UO126 and LY294002 were purchased from Cell Signaling Technology (Boston, MA, USA). Fasudil was purchased from Abcam (Cambridge, MA, USA) and the leptin-neutralizing antibody was purchased from R&D Systems (Minneapolis, MN, USA).
Kato S., Abarzua-Catalan L., Trigo C., Delpiano A., Sanhueza C., García K., Ibañez C., Hormazábal K., Diaz D., Brañes J., Castellón E., Bravo E., Owen G, & Cuello M. (2015). Leptin stimulates migration and invasion and maintains cancer stem-like properties in ovarian cancer cells: an explanation for poor outcomes in obese women. Oncotarget, 6(25), 21100-21119.
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8

Inhibiting PI3K/AKT/mTOR and ERK1/2 in VSMC

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We applied LY3023414 (Ly30; Selleckchem, Munich, Germany), a PI3K/AKT/mTOR inhibitor, to interfere with EVUR‐induced activation of AKT in VSMC. The activation of ERK1/2 in VSMC was blocked by applying the inhibitor UO126 (Cell signalling). In Western blot experiments, the cells were pre‐incubated with 50 nmol/L Ly30 or 10 µmol/L UO126 for 5 minutes prior to the respective treatment. Both inhibitors were dissolved in DMSO and the resulting maximum DMSO concentration in the experiments was 0.05%. To avoid long‐term toxicity of the inhibitors, the VSMC were treated prior to every medium change (every 2nd day) for 5 minutes with Ly30 or UO126.
Freise C., Querfeld U., Ludwig A., Hamm B., Schnorr J, & Taupitz M. (2021). Uraemic extracellular vesicles augment osteogenic transdifferentiation of vascular smooth muscle cells via enhanced AKT signalling and PiT‐1 expression. Journal of Cellular and Molecular Medicine, 25(12), 5602-5614.
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9

Murine Macrophage Activation by Biglycan

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Murine thioglycolate-elicited macrophages were isolated from peritoneal lavage and grown in RPMI 1640 (Life Technologies, Germany) supplemented with 1% penicillin and streptomycin and 2% fetal bovine serum (Biochrom, Germany). Cells were stimulated with 4 μg/ml (80 nM) human biglycan in serum-free medium for indicated time periods. Phorbol 12-myristate 13-acetate (PMA) (100 nM; Sigma, Germany) served as positive control for p47phox translocation studies. For ROS inhibition diphenyleneiodonium chloride (DPI, 0.5 μM; Sigma, Germany), VAS2870 (5 μM; Sigma, Germany), ML-171 (10 nM; Merck, Germany), Nox2ds-tat (40 μM; AnaSpec, USA) and GKT137831 (200 μM) were applied to the macrophages 1 h prior to stimulation with biglycan. For immunofluorescence studies Akt inhibitor 124008 (1 μM; Calbiochem, Germany), p38 MAPK inhibitor SB203580 (10 μM; Calbiochem, Germany), MEK1/2 inhibitor UO126 (10 μM; Cell Signaling, Germany), PI3K inhibitor LY294002 (10 μM; Cell Signaling, Germany), Rac1 inhibitor (50 μM; Calbiochem, Germany), PKC inhibitor GO6976 (20 nM; Sigma, Germany) and MyD88 inhibitor peptide NBP2–29328 (50 μM; Novus Biologicals, Germany) were applied 1 h prior to stimulation with biglycan. For HSP70 inhibition phenylsulfonamide (PES, pifithrin-μ) (200 μM; Sigma, Germany) was applied to the cells in addition to biglycan.
Hsieh L.T., Frey H., Nastase M.V., Tredup C., Hoffmann A., Poluzzi C., Zeng-Brouwers J., Manon-Jensen T., Schröder K., Brandes R.P., Iozzo R.V, & Schaefer L. (2015). Bimodal role of NADPH oxidases in the regulation of biglycan-triggered IL-1β synthesis. Matrix biology : journal of the International Society for Matrix Biology, 49, 61-81.
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10

Modulation of PGE2 Production

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SC (1 × 105/mL, 12-well plates) were pre-treated for 2 h with 10 µM COX-2 inhibitor (CAS 181696-33-3, Calbiochem, San Diego, CA, USA), 10 µM TGF-βR1/ALK4/5/7 inhibitor (SB431542, MilliporeSigma), 10 µM MAPK/ERK inhibitor (UO126, Cell Signaling Technology) or medium served as a control. Medium was removed and SC were treated with tumor-conditioned medium (10% v/v), control medium or rTGF-β1 (100 pg/mL, Cell Signaling Technology, Danvers, MA, USA) for 24 h. To block the TGF-β1 activity, 10 μg/mL anti-TGF-β1-neutralizing antibody (R&D System, Minneapolis, MN, USA) was added at the beginning of stimulation. Medium was removed and replaced with complete medium (2% FBS) for additional 24-h incubation. Cell-free supernatants were collected by centrifugation at 700× g for 5 min. PGE2 concentration (Cayman Chemical, USA) was measured in supernatants by ELISA.
Shurin G.V., Vats K., Kruglov O., Bunimovich Y.L, & Shurin M.R. (2022). Tumor-Induced T Cell Polarization by Schwann Cells. Cells, 11(22), 3541.
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