Ly2090314

Manufactured by Selleck Chemicals

Sourced in United States

LY2090314 is a chemical compound used in laboratory research. It functions as a small molecule inhibitor. The core purpose of this product is to serve as a research tool for scientific investigations.

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Lab products found in correlation

8 protocols using ly2090314

Small Molecule Inhibitor Screening

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CHIR99021 (S1263, Selleckchem, distributed by LuBioScience, Zurich, Switzerland) was used at different concentrations. LY2090314 (S7063, Selleckchem) was used at 100 nM. AZD1080 (S7145, Selleckchem) was used at 10 µM. Vemurafenib (PLX4032, RG7204), (S1267, Selleckchem) was used at 1 μM. Dabrafenib (S2807, Sellechchem) was used at 1 μM. Selumetinib (AZD6244), (S1008, Selleckchem) was used at 1 μM MG-132, (S2619, Selleckchem) was used at 20 μM.

Uka R., Britschgi C., Krättli A., Matter C., Mihic D., Okoniewski M.J., Gualandi M., Stupp R., Cinelli P., Dummer R., Levesque M.P, & Shakhova O. (2020). Temporal activation of WNT/β-catenin signaling is sufficient to inhibit SOX10 expression and block melanoma growth. Oncogene, 39(20), 4132-4154.

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Extracellular Matrix Modulates hTM Cell Mechanics

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Vehicle control CDM and XCDMs (on glass coverslips or dishes) obtained were primed with serum-free media at room temperature for approximately 4 hours. After removing the media, low passage hTM cells from the same donor used to derive ECMs were seeded (5,000–10,000 per cm²) on CDM or 1%, 2%, and/or 10% XCDMs in serum-free media for 24 hours. In other parallel experiments, hTM cells were cultured on CDM or 10% XCDM in serum-free media for 24 hours, with or without 250 nM Wnt signaling activator, LY2090314 (Selleckchem, Houston, TX, USA) or 10 µM Wnt signaling inhibitor, LGK974 (Selleckchem, Houston, TX, USA). Subsequently, in subsets of these experiments, cell mechanics was determined via AFM, RNA was extracted for reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and protein was extracted from whole cell lysates/subcellular fractions for Western blotting.

Yemanyi F., Vranka J, & Raghunathan V.K. (2020). Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways. Investigative Ophthalmology & Visual Science, 61(10), 41.

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Wnt Signaling Effects on Mesenchymal Cells

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Suture mesenchymal cells from one-month-old C57BL/6J mice were cultured with 20 μM Wnt agonist 1 (Selleck, S8178) or 100 nM LY2090314 (Selleck, S7063) for one or two weeks in αMEM. Medium was changed every other day. Total protein was extracted using a solution of loading buffer (Cell Signaling Technology, 7723S), protease inhibitor (Thermo Fisher Scientific, 1861278) and DTT (Cell Signaling Technology, 7723S), then separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, ISEQ00005). Membranes were blocked with 5% non-fat dry milk dissolved in TBST for 2 hours at room temperature with gentle shaking, and then incubated with primary antibodies: anti-Runx2 (Cell signaling technology 12556, 1:1000), anti-OPN (Abcam ab63856, 1:500), and anti-βactin (Abcam ab20272, 1:1000) at 4 ° overnight followed by corresponding horseradish-peroxidase (HRP)-conjugated secondary antibodies. Protein expression was detected by Bio-Rad ChemiDoc Touch (Bio-Rad) and intensities of bands were quantitated by Image J software.

Yu M., Ma L., Yuan Y., Ye X., Montagne A., He J., Ho T.V., Wu Y., Zhao Z., Maria N.S., Jacobs R., Urata M., Wang H., Zlokovic B.V., Chen J.F, & Chai Y. (2021). Cranial suture regeneration mitigates skull and neurocognitive defects in craniosynostosis. Cell, 184(1), 243-256.e18.

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Wnt5A Signaling, Autophagy, and GSK3 Inhibition

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Cells were treated with 100ng/mL or 200ng/mL of recombinant Wnt5A (rWnt5A, R&D Systems, cat. no. 645WN010CF) for 16 hours. Treatment with bafilomycin A1 (Sigma, cat. no. B1793) was performed at a final concentration of 100nM for 3–4 hours. The autophagy inhibitor Lys05 (obtained from the laboratory of Dr. Ravi Amaravadi) was used at various concentrations in vitro (1, 3, 5, and 10μM) for 16–24 hours and in vivo in mice at 20mg/kg daily for 14 days total. Cells were treated with the GSK3 inhibitor, lithium chloride (Sigma, cat. no. 203637), at various concentrations (2 mM, 5 mM and 10 mM) for 8 hours. Treatment with the GSK3 inhibitor, LY2090314 (Selleckchem cat. no. S7063) was performed at various concentrations (2, 5, and 10 nM) for 8 hours. For combination treatments, cells were first treated with rWnt5A for 16 hours, then bafilomycin A1 was added for 3 hours or Lys05 for 16 hours. For treatments combining GSK3 inhibitors with Lys05, cells were first treated with lithium chloride or LY2090314 for 8 hours followed by Lys05 treatment for 16 hours.

Ndoye A., Budina-Kolomets A., Kugel CH I.I.I., Webster M., Kaur A., Behera R., Rebecca V., Li L., Brafford P., Liu Q., Gopal Y.N., Davies M.A., Mills G.B., Xu X., Wu H., Herlyn M., Nicastri M., Winkler J., Soengas M.S., Amaravadi R., Murphy M, & Weeraratna A.T. (2017). ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma. Cancer research, 77(21), 5873-5885.

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Genetic manipulation of GSK-3β in cell lines

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All cell lines (HeLa, MDA-MB-231, and HEK293T) were obtained from the American Type Culture Collection and were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, 8121298) supplemented with 10% fetal bovine serum (FBS, Gibco, 2176398) and 1% penicillin–streptomycin at 37 °C with 5% CO₂.
The following regents were used in this study: Erastin (E7781), Fer-1 (S7243), and LY2090314 (S7063) were purchased from Selleck (Houston, TX, USA). The coding region of GSK-3β gene was cloned into the pCMV vector in-frame with a myc tag within the vector. The pLKO.1-puro shRNA vector was obtained from Sigma-Aldrich (SHC001). The pLKO.1-shGSK-3β 1^# and 2^# were constructed using specific primer containing separate targeting sequence. (targeting sequence to GSK-3β 1^#: 5′-CACTGGTCACGTTTGGAAAGA-3′, GSK-3β 2^#: 5′-CCCAAACTACACAGAATTTAA-3′).

Wang L., Ouyang S., Li B., Wu H, & Wang F. (2021). GSK-3β manipulates ferroptosis sensitivity by dominating iron homeostasis. Cell Death Discovery, 7, 334.

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Modulating Pancreatic Cancer Metastasis

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Human pancreatic cancer cell lines (BxPC-3, PANC-1, and CFPAC-1) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). Pancreatic cancer cells were serum-starved for 12 h before treatment with 10 µM LPA (Sigma-Aldrich) for another 24 h and tested for gene expression, migration, and invasion. TGF-β1 recombinant protein (50 ng/mL; PeproTech, Rocky Hill, NJ, USA) was also used to induce pancreatic cancer cell migration and invasion. In some experiments, pancreatic cancer cells were treated with LY2090314 (50 nM; Selleckchem, Houston, TX, USA) for 24 h before transfection with CMTM8 shRNA.

Shi W., Zhang C., Ning Z., Hua Y., Li Y., Chen L., Liu L., Chen Z, & Meng Z. (2021). CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis. Annals of Translational Medicine, 9(1), 42.

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Investigating Retinal Protein Interactions

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TASC (MAB19294) and W1B10 (I8638) were purchased from Millipore and Sigma, respectively. A monoclonal mouse IgG1 isotype control antibody M075-3M2 (Functional Grade) was purchased from MBL (Nagoya, Japan). TASC was applied at 20, 50, 100, 200 μg/mL. W1B10 was applied at 10, 20, 50, 100, 200 μg/mL. M075-3M2 was applied at 100, 200 μg/mL. Retinal strips were preincubated in a drop of DMEM (25 μL) containing the antibody on Parafilm® for 10 min. Since the antibodies were presented in phosphate buffer, decreases in free Ca²⁺ and Mg²⁺ due to the phosphate buffer were compensated by adding CaCl₂ and MgCl₂ to the preincubation medium and Matrigel® at the ratio of 4 (phosphate buffer): 2 (CaCl₂): 1 (MgCl₂). LY2090314 (Selleck) was dissolved in DMSO at 10 mM as a stock solution and diluted to 1 mM for an aliquot of 25 μL. SC79 (Selleck) was dissolved in DMSO at 25 mM as a stock solution. Omipalisib (GSK2126458, GSK458, Selleck) was dissolved in DMSO at 5 mM as a stock solution. Cytochalasin D (Cayman Chemical) was dissolved in DMSO at 1 mg/mL as a stock solution. Calbryte^TM−520L potassium salt (20642, AAT Bioquest®) was dissolved in distilled water at 10 mM containing 1 mM CaCl₂. Nocodazole (FUJIFILM Wako) was dissolved in DMSO at 10 mg/mL as a stock solution.

, & Yamashita M. (2023). Integrin-mediated electric axon guidance underlying optic nerve formation in the embryonic chick retina. Communications Biology, 6, 680.

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Transcriptional Reporter Plasmid Construction

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The protein kinase inhibitors Torin 1, rapamycin, LY2090314, and CHIR99021 were purchased from Selleckchem (Houston, Texas, USA), whereas PTEN (sc-29459) and negative control (sc-37007) siRNAs were from Santa Cruz Biotechnology, Inc. (Dallas, Tex.). All other chemicals were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). The LUC reporter plasmid MT1-LUC contains 1843 bp of the 5= flanking region and 68 bp of the 5= untranslated region from the mouse Mt1 gene (Faraonio et al. 2000) (link). Similarly, the plasmid MT2A-LUC contains a human Mt2A gene DNA fragment (positions -780 to +65) cloned into pGL2 basic (Promega, Madison, Wisconsin, USA) (Dubé et al. 2011 (link)). To construct plasmid (MREd) 6 -LUC, a synthetic DNA fragment containing 6 mouse Mt1 MREd elements (Labbé et al. 1991) (link) (5 elements in direct tandem orientation and the 6th in the opposite orientation) was cloned in front of a minimal mouse Mt1 promoter DNA fragment (positions -35 to -68) into the LUC reporter plasmid pGL2-Basic (Promega, Madison, Wis.). TOP-Flash and FOP-Flash plasmids were purchased from Upstate (Temecula, California, USA).

Andéol Y., Bonneau J., M Gagné L., Jacquet K., Rivest V., Huot M.É, & Séguin C. (2018). The phosphoinositide 3-kinase pathway and glycogen synthase kinase-3 positively regulate the activity of metal-responsive transcription factor-1 in response to zinc ions. Biochemistry and cell biology = Biochimie et biologie cellulaire.

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