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Ku 0063794

Manufactured by Merck Group
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KU-0063794 is a chemical compound developed by Merck Group. It is a laboratory tool used for research purposes. The core function of KU-0063794 is to serve as a research tool for scientific investigations, without further interpretation of its intended use.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Vegf a, by Thermo Fisher Scientific (1 mentions) Fitc phalloidin, by Merck Group (1 mentions) Anti phospho akt s473, by Cell Signaling Technology (1 mentions) Anti phospho fak y397, by Cell Signaling Technology (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Endothelial cell growth supplement (ecgs), by Thermo Fisher Scientific (1 mentions) Anti vinculin, by Merck Group (1 mentions) Anti fak, by Cell Signaling Technology (1 mentions) Anti s6k, by Abcam (1 mentions) Anti akt1, by Proteintech (1 mentions) Anti phospho s6k t389, by Cell Signaling Technology (1 mentions) Anti raptor, by Cell Signaling Technology (1 mentions) Anti rictor, by Cell Signaling Technology (1 mentions) Effectene transfection reagent, by Qiagen (1 mentions) Fk506, by Merck Group (1 mentions) Rapamycin, by Cell Signaling Technology (1 mentions) Nvp bez235, by Selleck Chemicals (1 mentions) Mln0128, by Selleck Chemicals (1 mentions)

8 protocols using ku 0063794

1

mTOR Modulation Impacts Endothelial Angiogenesis

Cited 2 times
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Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen (Burlington, ON). VEGF-A was from Peprotech (Princeton, NJ). Rapamycin, PP242, a highly specific mTOR active-site inhibitor [12 (link)], and anti-tubulin-α was from Millipore (Temecula, CA). Ku-0063794, a second specific mTOR inhibitor [13 (link)] was from Sigma (St. Louis, MO). Anti-Akt1 was from Protein Tech (Chicago, IL). Hiperfect, non-silencing short interfering RNA (siRNA) and Akt1 silencing siRNA were from Qiagen Inc (Mississauga, ON). Human tumor necrosis factor-α (TNFα) was from Cedarlane (Mississauga, ON). Cycloheximide, phalloidin-FITC, anti-vinculin, and DAPI were from Sigma. Anti-S6K was from Abcam (Cambridge, UK). Anti-phospho-AktS473, anti-phospho-S6KT389, anti-FAK, anti-phospho-FAKY397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology (Danvers, MA). ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific (Waltham, MA).
Farhan M.A., Carmine-Simmen K., Lewis J.D., Moore R.B, & Murray A.G. (2015). Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis. PLoS ONE, 10(8), e0135245.
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2

Glioma Cell Line Transfection and Transduction

Cited 1 time
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Human glioma cell lines were grown in 10% FBS. These included LN229, U87MG, GBM43, GBM5, and GBM12 (Sarkaria et al., 2006 (link)). Plasmids pcDNA3-mTORWT, pcDNA3-mTORR2505P, pcDNA3-mTORS2215Y (Sato et al., 2010 (link)) were obtained from Addgene (plasmids #26036-8); and transfected stably into LN229 cells using Effectene-transfection reagent (Qiagen). To generate retrovirus to transduce LN229 and U87MG with EGFR or EGFRvIII (Fan et al., 2007 (link)), the packaging cell line 293T was co-transfected with pWLZ-hygro-EGFR plasmid gag/pol and VSVg or with pLRNL-neo-EGFRvIII plasmid gag/pol and VSVg again using Effectene. High-titer virus was collected at 48 hr and used to infect cells as described (Fan et al., 2006 (link)). Transfected and transduced cells were selected as pools with G418 (800 μg/ml) or hygromycin (500 μg/ml) for two weeks. Inhibitors INK1437, TGX221, IC87114, INK1358, AS252424, AS605240, and GDC-0941 were from Pingda Ren and Liansheng Li. AKT inhibitor VIII was from EMD Biosciences. Rapamycin was from Cell Signaling. NVP-BEZ235, MLN0128, and MK-2206 were from Selleck Chemicals. KU-0063794 and FK-506 were from Sigma-Aldrich Inc. Insulin was from Eli Lilly & Co. D-luciferin was from Gold Biotechnology USA. RapaLink-1 and RapaLink-2 were synthesized by CM, MO, and KMS as described (Rodrik-Outmezguine et al., 2016 (link)).
Fan Q., Aksoy O., Wong R.A., Ilkhanizadeh S., Novotny C.J., Gustafson W.C., Truong A.Y., Cavanan G., Simonds E.F., Haas-Kogan D., Phillips J.J., Nicolaides T., Okaniwa M., Shokat K.M, & Weiss W.A. (2017). A Kinase Inhibitor Targeted to mTORC1 Drives Regression in Glioblastoma. Cancer cell, 31(3), 424-435.
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3

Viability Evaluation of Compound Treatments

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Compound treatments were performed in 96-well clear plates (Greiner, 353072) and 96-well black plates to evaluate viability (Greiner, 655090). HEK293T cells (15,000 cells/well) were treated 24 h post seeding, while rat neurons and astrocytes were treated at 4 DIV post seeding (50,000 and 3,000 cells/well respectively). All tested compounds KU0063794 (Sigma-Aldrich, SML0382), NVP-TAE684, SU11652 and bafilomycin A1 (Sigma-Aldrich, B1793) were dissolved in DMSO and serially diluted for testing. The final amount of DMSO solutions tested in cell media never exceeded 0.25% v/v. 2, 6 or 24 hours post-treatment cells were PBS washed and lysed in 40 μl of lysis buffer per well: TBS, 0.4% Triton- X, protease inhibitors cocktail (Roche, 11836170001).
Bresciani A., Spiezia M.C., Boggio R., Cariulo C., Nordheim A., Altobelli R., Kuhlbrodt K., Dominguez C., Munoz-Sanjuan I., Wityak J., Fodale V., Marchionini D.M, & Weiss A. (2018). Quantifying autophagy using novel LC3B and p62 TR-FRET assays. PLoS ONE, 13(3), e0194423.
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4

Autophagy Modulation in Cell Lines

Cited 1 time
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MelJuSo Ub-YFP cells have been previously described [24 (link)]. HOS parental and GFP-LC3B were obtained from Gerald McInerney [58 (link)]. U2OS mRFP-GFP-LC3 were obtained from Jeff MacKeigan [59 (link)]. HeLa TREx FlpIn WT were a kind gift from A. Tighe and S.S. Taylor (University of Manchester, UK) and used to generate the ATG13 and ATG16L1 knockout cell lines, as described below. HEK293 WT cells were obtained from Tamotsu Yoshimori (Osaka University, Japan) and were used to generate ATG16L1 knockout cell lines reconstituted with either eGFP-ATG16L1β or eGFP-ATG16L1[1–249] as described in [40 (link)]. All cells were cultured in DMEM+GlutaMAX (Life Technologies, 31,966–021) supplemented with 10% fetal bovine serum (Life Technologies, 10,270–106) in a humidified incubator at 37°C and 5% CO2. All cell lines were routinely tested for Mycoplasma infection.
To induce autophagy, cells were treated with 250 nM Torin 1 (Tocris, 4247) or with 500 nM KU-0063794 (Sigma-Aldrich, SML0382). Autophagy was blocked using 10 mM 3-methyladenine (3-MA; Sigma-Aldrich, 189,490), 100 nM bafilomycin A1 (BafA1; Enzo Life Sciences, BML-CM110) or 10 µM chloroquine (CQ; Sigma-Aldrich, C6628).
Giovannucci T.A., Salomons F.A., Stoy H., Herzog L.K., Xu S., Qian W., Merino L.G., Gierisch M.E., Haraldsson M., Lystad A.H., Uvell H., Simonsen A., Gustavsson A.L., Vallin M, & Dantuma N.P. (2021). Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy. Autophagy, 18(7), 1486-1502.
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5

Endothelial Cell Signaling Pathway Regulation

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FSH (#F4021), 17β-estradiol (E2, #E2758), TNF-α(#T0157), pertussis toxin (PTX, #3097), β-methyl cyclodextrin (β-MCD, #C-4555), and KU0063794 (#SML0382) were purchased from Sigma Aldrich (St. Louis, MO, USA). 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3,4-d) pyrimidine (PP2, #1407), pyrrolidine dithiocarbamate (PDTC, #0727), PD98059 (#1213), NF499 (#1391), forskolin (#1099), MDL12330A(#1436) were obtained from Tocris Bioscience (Minneapolis, MN, USA). LY294002 (LY, #9901s) and H89 (#9844s) were acquired from Cell Signaling Technologies (CST, Beverly, MA, USA). Endothelial Basal Medium-2 (EBM-2) (#190860) was purchased from Lonza (Walkersville, MD, USA). Opti-MEM (#31985) and fetal bovine serum (FBS, #12484) were obtained from Invitrogen (Carlsbad, CA, USA). All other chemicals were of analytical grade and purchased from Guangzhou Chemical Reagents (Guangzhou, China).
Li X., Chen W., Li P., Wei J., Cheng Y., Liu P., Yan Q., Xu X., Cui Y., Gu Z., Simoncini T, & Fu X. (2017). Follicular Stimulating Hormone Accelerates Atherogenesis by Increasing Endothelial VCAM-1 Expression. Theranostics, 7(19), 4671-4688.
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6

Hedgehog Signaling Modulator Assay

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Smoothened agonist SAG was purchased from Calbiochem. SANT (SANT-1); KU-0063794, GSK-690693, PF-4708671 and MG132 were from Sigma and AZ191 was from SelleckChem. Cycloheximide was purchased from Biomol.
The monoclonal Hedgehog neutralizing antibody (5E1, supernatant), developed/deposited by T.M. Jessell/S. Brenner-Morton was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.
Singh R., Dhanyamraju P.K, & Lauth M. (2016). DYRK1B blocks canonical and promotes non-canonical Hedgehog signaling through activation of the mTOR/AKT pathway. Oncotarget, 8(1), 833-845.
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7

Rapamycin, Ku0063794, and DMSO Protocols

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Rapamycin, Ku0063794, and DMSO were purchased from Sigma.
Lu Q., Liu Y., Wang Y., Wang W., Yang Z., Li T., Tian Y., Chen P., Ma K., Jia Z, & Zhou C. (2017). Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells. Bioscience Reports, 37(3), BSR20160552.
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8

Metabolic Modulation in Jurkat T Cells

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Jurkat T cells were cultured in R10FBS supplemented with 2 mM glutamine. Chemicals and inhibitors used in this study were as follows: 2-DG (10 mM), Akti1/2 (10 μM), Osi-027 (10 μM), KU-0063794 (10 μM), Rotenone (1 μM), TTFA (200 μM), Antimycin A (2 μM), Oligomycin (0.1 or 1 μM) or 4-(trifluoromethoxy) phenylhydrazone (FCCP, 2 μM), Clotrimazol (25 μM), nocodazole (10 μM), oxamate (5 mM), methyl pyruvate (1 or 10 mM), bifonazole (25 µM), MB-3 (1, 10 µM), 2-NBDG (0.5 mM) and mitotempo (20 μM) (all from Sigma-Aldrich). TCS-2002 (10 µM), SB 204990 (3 µM) and garcinol (1, 10 µM) (Tocris) was used at 10 μM. VDAC peptides corresponding to loop 4 (LP4 - KKLETAVNLAWTAGNSN) flanked N-, and C-terminally by the tryptophan zipper motif for increased stability (SWTWE and KWTWK, respectively) were linked to tandem repeats of the DNP2 fusion peptide (KIKKVKKKGRKKIKKVKKKGRK). The complete sequence for the peptides used are: dNP2 (KIKKVKKKGRKKIKKVKKKGRKSWTWEKWTWK) and dNP2-VDAC (KIKKVKKKGRKKIKKVKKKGRKSWTWEKKLETAVNLAWTAGNSNKWTWK). Both peptides were synthesized by Biomatik.
Bantug G.R., Fischer M.G., Grählert J., Balmer M.L., Unterstab G., Develioglu L., Steiner R., Zhang L., da Costa A.S., Gubser P.M., Burgener A.V., Sauder U., Löliger J., Belle R., Dimeloe S., Lötscher J., Jauch A., Recher M., Hönger G., Hall M.N., Romero P., Frezza C, & Hess C. (2018). Mitochondria–ER contact sites are immunometabolic hubs that orchestrate the rapid recall response of memory CD8+ T cells. Immunity, 48(3), 542-555.e6.
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