Nvp aew541

Manufactured by Selleck Chemicals

Sourced in United States

NVP-AEW541 is a chemical compound used as a lab equipment product. It serves as a core function in various research and analytical applications.

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AEW541 (6 citations)

24 protocols using nvp aew541

Evaluating NVP-AEW541 Effects on Wilt49 Cells

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To assess the effect of NVP-AEW451 on cell viability, parental and PLAG1-overexpressing WiT49 cells were seeded in four replicate wells of a 96-well plate at 3 × 10³ cells per well. The next day, medium containing NVP-AEW541 (Selleck Chemicals) was added at concentrations ranging from 125 µM to 2 nM. After 72 h, the number of viable cells was quantified using CellTiter-Glo 2.0 (Promega). The IC50 was calculated using a nonlinear fit with variable slope (GraphPad Prism). Data are presented as IC50 in micromolar (95% confidence interval).
To assess the effect of NVP-AEW451 on S6 phosphorylation, parental WiT49- and PLAG1L-overexpressing cells were each seeded in three wells of a six-well plate at 2.5 × 10⁵ cells per well. The next day, cells were treated with either 0.02% DMSO for 6 h or 5 µM NVP-AEW541 (Selleck Chemicals) for 3 or 6 h. The cells were then harvested for protein lysate and used for immunoblotting.

Chen K.S., Stroup E.K., Budhipramono A., Rakheja D., Nichols-Vinueza D., Xu L., Stuart S.H., Shukla A.A., Fraire C., Mendell J.T, & Amatruda J.F. (2018). Mutations in microRNA processing genes in Wilms tumors derepress the IGF2 regulator PLAG1. Genes & Development, 32(15-16), 996-1007.

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Characterization of HNSCC Cell Lines

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The HNSCC cell lines (SCC9, SCC15, CAL27, SCC25 and MDA1386) were obtained, characterized, grown in media and condition as previously described (24 ). All of the cell lines have been authenticated by short tandem repeat profiling within six months of passage. The phospho-receptor tyrosine kinase array was purchased (ARY001B, R&D Systems, Minneapolis, MN). The array layout of the 49 RTKs were shown in the Supplemental Figure (Fig. S5). The following small molecular TKIs were purchased (Selleck Chemicals): (1) JNJ-38877605, a highly selective, ATP-competitive inhibitor of c-MET (25 (link)); (2) NVP-AEW541, a potent inhibitor of IGF-1R with IC₅₀ of 86 nM (26 (link)); (3) OSI-744/erlotinib HCl, a FDA approved EGFR inhibitor and (4) STI-571/imatinib, a multi-target inhibitor of v-Abl, c-Kit and PDGFR. The TKIs were reconstituted in DMSO solvent as per manufacture recommendation.

Klinghammer K., Keller J., George J., Hoffmann J., Chan E.L, & Hayman M.J. (2017). A phosphoarray platform is capable of personalizing kinase inhibitor therapy in head and neck cancers. International journal of cancer, 142(1), 156-164.

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Adipogenic Differentiation of hMSCs

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hMSCs were seeded into four-well plates and exposed to adipogenic induction media composed of DMEM, 10% fetal bovine serum (FBS), 10% horse serum (Gibco, U.S.A.), 100 µM dexamethasone (Sigma, U.K.), 1 µM Rosiglitazone (BRL) (Novo Nordisk Bagsvaerd, Denmark), 3 µg/ml insulin (Sigma, U.K.), 450 µM isobutylmethylxanthine (IBMX) (Sigma, U.K.), and 1% penicillin-streptomycin (Sigma, U.K.) supplemented with FAK inhibitors (PF-573228 and PF-562271) or IGF-1R/InsR inhibitors (NVP-AEW541 and GSK1904529A), which were purchased from Selleckchem Inc. (Selleckchem Inc., Houston, TX, U.S.A.). Inhibitors were used at 5 µM throughout all experiments. Adipocyte induction medium (AIM) was changed every 2 days and for 7 days. Previous published work from our group indicated day 7 as a time point on which adipogenic markers were up-regulated significantly in an enriched population of 70–80% of adipogenic populations [8 (link)].

Ali D., Abuelreich S., Alkeraishan N., Shwish N.B., Hamam R., Kassem M., Alfayez M., Aldahmash A, & Alajez N.M. (2018). Multiple intracellular signaling pathways orchestrate adipocytic differentiation of human bone marrow stromal stem cells. Bioscience Reports, 38(1), BSR20171252.

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Inhibitor Screening and Cell Treatment

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Imatinib (1 μM) was purchased from Tocris Bioscience. BKM-120 (5 μM) was purchased from Active Biochemical Co. dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich. NVP-AEW541 (5 μM) was purchased from Selleck. Bortezomib (50 nM) was purchased from LC Laboratories, GSK11220212 (250 nM) was provided by the Cantley lab (BIDMC). Cells in normal growth conditions, 70% confluence, were treated with inhibitors for one hour prior to lysis using DMSO as vehicle.
PHA-665752 was purchased from Tocris. Gefitinib (1 μmol/L) were obtained from American Custom Chemical. TAE-684 (100 nmol/L) was purchased from Selleck. Rapamycin (50 nmol/L) was purchased from Sigma. Cells in normal growth conditions, 70% confluence, were treated with inhibitors for 6 hour, except for Rapamycin, which was used for 16 hours prior to lysis using DMSO as vehicle.

Breitkopf S.B., Yang X., Begley M.J., Kulkarni M., Chiu Y.H., Turke A.B., Lauriol J., Yuan M., Qi J., Engelman J.A., Hong P., Kontaridis M.I., Cantley L.C., Perrimon N, & Asara J.M. (2016). A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2. Scientific Reports, 6, 20471.

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Investigating EGFR and IGF-1R Inhibitors in Lung Cancer Cell Lines

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The cell lines NCI-H460 and NCI-H1975 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), and incubated at 37°C, 5% CO₂. The EGFR inhibitor Gefitinib (#4765) was purchased from Cell Signaling Technology. The IGF-1R inhibitor NVP-AEW541 (#S1034) was purchased from Selleckchem. RIPA lysis and extraction buffer (#89900) used to lyse cells was purchased from ThermoFisher. PIK3R2-shRNA (#sc-39125-SH) and the Control shRNA Plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology. MiR-30a-5p mimics (5′-UGUAAACAUCCUCGACUGGAAG-3′) and the negative control RNA oligo (5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from GenePharma. PIK3R2-shRNA and miR-30a-5p mimics were transfected by lipofectamine 2000 reagent (#12566014) purchased from ThermoFisher. Annexin V-FITC Apoptosis Detection Kit (#K101-25) was purchased from BioVision. The CytoSelect™ Cell Invasion Assay Kit (#CBA-110) was purchased from CELL BIOLABS, INC.

Meng F., Wang F., Wang L., Wong S.C., Cho W.C, & Chan L.W. (2016). MiR-30a-5p Overexpression May Overcome EGFR-Inhibitor Resistance through Regulating PI3K/AKT Signaling Pathway in Non-small Cell Lung Cancer Cell Lines. Frontiers in Genetics, 7, 197.

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Preliminary Screening of Toxoplasma Inhibitors

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The Selleck New Compound Library, consisting of 666 compounds (each stored as a 10 mM stock solution in DMSO), was obtained from the Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences, China (http://www.sibcb.ac.cn/cp13-5_3.asp), and used for preliminary screens against Toxoplasma. For further studies, both NVP-AEW541 and GSK-J4 HCl were obtained from Selleck (Cat# S7070 and S1034, respectively). Pyrimethamine (Cat# 46706, Sigma-Aldrich, Shanghai, China) was included in the experiments as a reference drug. A stock solution of these compounds was prepared in 100% dimethyl sulfoxide (DMSO, Sigma-Aldrich), stored at − 80 °C, and diluted in fresh culture medium prior to each use, with a final DMSO concentration below 0.1%. A23187 (Cat# 100105, Sigma-Aldrich) was dissolved in DMSO at 1 mM and stored at − 20 °C.

Liu S., Wu M., Hua Q., Lu D., Tian Y., Yu H., Cheng L., Chen Y., Cao J., Hu X, & Tan F. (2020). Two old drugs, NVP-AEW541 and GSK-J4, repurposed against the Toxoplasma gondii RH strain. Parasites & Vectors, 13, 242.

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Western Blotting with Targeted Inhibitors

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For western blotting, the following antibodies were used: phospho-EGFR Y1068 (Abcam); EGFR, pERK1/2 T202/204, ERK1/2, p-AKT S473, BIM, Actin (Cell Signaling); AKT1/2/3 (Santa Cruz). For cell culture studies, gefitinib, WZ4002, NVP-AEW541, ABT-263 (all from Selleck), were dissolved in DMSO to a final concentration of 10 mmol/l and stored at −20°C. Unless otherwise specified, 1 μM concentration was used for in vitro cell culture experiments.

Hata A.N., Niederst M.J., Archibald H.L., Gomez-Caraballo M., Siddiqui F.M., Mulvey H.E., Maruvka Y.E., Ji F., Bhang H.E., Radhakrishna V.K., Siravegna G., Hu H., Raoof S., Lockerman E., Kalsy A., Lee D., Keating C.L., Ruddy D.A., Damon L.J., Crystal A.S., Costa C., Piotrowska Z., Bardelli A., Iafrate A.J., Sadreyev R.I., Stegmeier F., Getz G., Sequist L.V., Faber A.C, & Engelman J.A. (2016). Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nature medicine, 22(3), 262-269.

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Flow Cytometry-Based Viability and Apoptosis

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For flow cytometry experiments, cells were plated and grown to 80% confluence in complete media. Cultures were then exposed to the IGF1R inhibitor NVP-AEW541 (8 μM, Selleckchem), IGF1R/IR inhibitor BMS-754807 (20 μM, Selleckchem), or AR inhibitor enzalutamide (20 μM, Selleckchem), in media containing phenol red-free RMPI 1640 media supplemented with 2% charcoal-dextran-stripped FBS (Gemini BioProducts, CA, USA) for 24 or 48 h. As per the manufacturer’s recommended protocol, cells were then removed and stained with 7-AAD and Annexin-V (Affymetrix, Inc., San Diego, CA, USA) for viability and apoptosis assays, respectively. Cells were examined using an LSR II Benchtop Analyzer (BD Biosciences, San Jose, CA, USA) with FlowJo Collector’s Edition software (FloJo, LLC, Ashland, OR, USA).

Hamilton N., Austin D., Márquez-Garbán D., Sanchez R., Chau B., Foos K., Wu Y., Vadgama J, & Pietras R. (2017). Receptors for Insulin-Like Growth Factor-2 and Androgens as Therapeutic Targets in Triple-Negative Breast Cancer. International Journal of Molecular Sciences, 18(11), 2305.

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Western Blotting with Targeted Inhibitors

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NVP-AEW541 Cell Viability Assay

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Cells were plated at 4000 cells per well in a 96‐well plate. The following day, media were replaced with media containing NVP‐AEW541 (Selleck Chemicals; Stratech Scientific, Newmarket, UK) using DMSO as a carrier control (0.1%). Cells were incubated for 72 h before being assayed for viability using the CellTiter Aqueous One Solution Cell Proliferation Assay (Promega) following the manufacturer's instructions. Absorbance at 490 nm was measured on an ELx800 Absorbance Microplate Reader (BioTek).

Selfe J., Goddard N.C., McIntyre A., Taylor K.R., Renshaw J., Popov S.D., Thway K., Summersgill B., Huddart R.A., Gilbert D.C, & Shipley J.M. (2018). IGF1R signalling in testicular germ cell tumour cells impacts on cell survival and acquired cisplatin resistance. The Journal of Pathology, 244(2), 242-253.

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