Azd8055

Manufactured by Selleck Chemicals

Sourced in United States, Germany, China

AZD8055 is a small molecule inhibitor that targets the serine/threonine protein kinase mTOR (mammalian target of rapamycin). It functions as an ATP-competitive inhibitor of mTOR, affecting both mTORC1 and mTORC2 complexes.

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Lab products found in correlation

97 protocols using azd8055

Xenograft Mouse Model for SCID Tumor Study

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Approximately 6–8 week-old female SCID mice (NOD.CB17-Prkdc;
Envigo RMS.Inc) were injected subcutaneously with 5 × 10⁶CAL62 or CAL62-splice cells grown to 70% confluence and resuspended in 50%
Matrigel (CORNING) into the right and left flanks, respectively. Treatments were
administered by oral gavage when tumor volume approached 200 mm³ as
estimated by measuring the length and width with calipers (width²× length × 0.52). Tumor-bearing mice were randomly assigned into 5
treatment arms: Controls (vehicle-4% DMSO in 30% PEG 300); AZD8055 (10 mg/kg);
Trametinib (0.75 mg/kg); JQ1 (40 mg/kg); AZD8055 + Trametinib and AZD8055 + JQ1
(all drugs were from Selleckchem). Mice were weighed at the start of treatment
and every second day during the treatment period. AZD8055 was dissolved in a
mixture of 4% DMSO and 30% PEG 300 (SIGMA), Trametinib in 4% DMSO in corn oil
and JQ1 in 2% DMSO, 30% PEG 300 and 5% Tween 80. Treatments were administered by
oral gavage in a volume of approximately 200 μL. Tumor volume was
measured every 2–3 days with calipers. After 21 days mice were humanely
killed, and dissected tumors flash frozen for subsequent protein isolation. All
animal experiments were performed in accordance with a protocol approved by the
Institutional Animal Care and Use of Committee (IACUC) of Memorial Sloan
Kettering Cancer Center.

Krishnamoorthy G.P., Davidson N.R., Leach S.D., Zhao Z., Lowe S.W., Lee G., Landa I., Nagarajah J., Saqcena M., Singh K., Wendel H.G., Dogan S., Tamarapu P.P., Blenis J., Ghossein R.A., Knauf J.A., Rätsch G, & Fagin J.A. (2018). EIF1AX and RAS mutations cooperate to drive thyroid tumorigenesis through ATF4 and c-MYC. Cancer discovery, 9(2), 264-281.

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Investigating Glioblastoma Stem Cell Signaling

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For AZD8055 (Selleckchem #S1555, US) and rapamycin (Selleckchem #S1039), US) treatment, in Western blot, U87 and U251 initiating cells were treated with AZD8055 (0, 0.1, 0.2, and 0.3 μM) for 48 h or treated with rapamycin (0, 1, 2, and 3 µM) for 24 h. In Immunofluorescence analysis, U87 and U251 initiating cells were treated with AZD8055 (0.3 μM) for 48 h and rapamycin (3 µM) for 24 h. For 3-MA (Selleckchem #S2767, US) treatment, in neurosphere formation assay, cell viability assay and in vitro limiting dilution assay, U87 and U251 initiating cells were treated with 0.5 mM 3-MA.

Tao Z., Li T., Ma H., Yang Y., Zhang C., Hai L., Liu P., Yuan F., Li J., Yi L., Tong L., Wang Y., Xie Y., Ming H., Yu S, & Yang X. (2018). Autophagy suppresses self-renewal ability and tumorigenicity of glioma-initiating cells and promotes Notch1 degradation. Cell Death & Disease, 9(11), 1063.

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Reh Xenograft Mouse Model with AZD-8055

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For the Reh xenograft models, Reh cells (1 × 10⁷) were injected into immune-deficient mice B-NDG (Biocytogen, China) through tail-vein injection. For AZD-8055 treatment, AZD-8055 (Selleck, USA) was dissolved in SBE-β-CD (Selleck, USA) or Captisol (Ligand, USA), with administration to mice twice daily by oral gavage starting from the 10^th day after tail-injection. The tumor burden of Reh xenografts was determined by the percentage of GFP-labeled Reh cells in total bone marrow cells, which is determined by flow cytometry after removing erythrocytes. All our animal experiments were approved by the Medical Ethical Committee of Shanghai Jiao Tong University and SCMC.

Xu Y., Fang H., Chen Y., Tang Y., Sun H., Kong Z., Yang F., Kirschner-Schwabe R., Zhu L., Toker A., Xiao N., Zhou B.B, & Li H. (2022). The KRAS-G12D mutation induces metabolic vulnerability in B-cell acute lymphoblastic leukemia. iScience, 25(3), 103881.

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Autophagy Induction in HEK293 and SHSY-5Y Cells

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The human embryonic kidney HEK293 cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Euroclone), while human neuroblastoma SHSY-5Y cell line in DMEM-F12 (1:1, Gibco) at 37 °C under 5% CO₂; both mediums were supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), 2mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco). The induction of autophagy by nutrients starvation was obtained by incubating HEK293 cells with Earle’s balanced salt solution (EBSS; Sigma-Aldrich) for 4 h and SHSY-5Y cells for 8 h. In the other case, autophagy stimulation was obtained by treating cells with 100 nM AZD8055 (AZD), an ATP-competitive inhibitor directly targeting the mTOR catalytic site (Selleckchem) [37 (link)], for 8 h. Both cells lines were also starved or treated with AZD8055 for 30 min, 1 h, 1.5 h, and 2 h, as indicated in figure legend.

Bordi M., De Cegli R., Testa B., Nixon R.A., Ballabio A, & Cecconi F. (2021). A gene toolbox for monitoring autophagy transcription. Cell Death & Disease, 12(11), 1044.

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Optimizing Drug-Induced Senescence Protocols

Cited 1 time

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All drugs used were reconstituted and stored as directed by the supplier (etoposide, 4-hydroxytamoxifen (4-OHT), mitomycin C, and CASIN—all from Sigma-Aldrich; AZD8055—Selleckchem). For routine drug treatment, cells were seeded in complete media and allowed to bed down overnight, before media were aspirated and replaced with drug-supplemented complete media. For drug treatment in co-culture assays, cells were seeded directly into drug-supplemented complete media. Optimum concentrations and dosing periods for induction of senescence without cell killing were selected according to previously published experiments [19 (link)–22 (link)].

Walters H.E, & Cox L.S. (2021). Intercellular Transfer of Mitochondria between Senescent Cells through Cytoskeleton-Supported Intercellular Bridges Requires mTOR and CDC42 Signalling. Oxidative Medicine and Cellular Longevity, 2021, 6697861.

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Hematopoietic Stem Cell Enrichment and Analysis

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KU-63794 was obtained from CalBiochem and AZD8055 was purchased from Selleckchem. Phycoerythrin (PE) Cy7-conjugated anti-Sca-1 (Clone E13-161.7, rat IgG2a), APC-conjugated anti-c-kit (Clone 2B8, rat IgG2b), and purified rat anti-CD 16/CD32 (Clone 2.4G2, Fcγ receptor blocker, rat IgG2b) were purchased from BD Pharmingen (San Diego, CA). Both mouse and human Hematopoietic Progenitor (Stem) Cell Enrichment Set-DM were purchased from BD Biosciences. Recombinant mouse thrombopoietin (TPO) was purchased from R&D Systems (Minneapolis, MN). The rabbit anti-phospho-p70 S6 kinase (p70S6K) monoclonal antibody and active (cleaved) caspase-3 antibodies were purchased from Cell Signaling. The Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody was purchased from Invitrogen (Carlsbad, CA).

Yang A., Xiao X., Zhao M., LaRue A.C., Schulte B.A, & Wang G.Y. (2015). Differential Reponses of Hematopoietic Stem and Progenitor Cells to mTOR Inhibition. Stem Cells International, 2015, 561404.

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Autophagy Regulation by Small Molecules

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MRT compounds were synthesized as described (21 (link)). AZD8055 was from Selleck Chemicals, and bafilomycin A1 was from Enzo Life Sciences. Rabbit anti-ULK1, anti-ATG5, and anti-ATG13 were from Sigma. Rabbit anti-phospho-Ser-757 ULK was from Cell Signaling Technology, and rabbit anti-phospho-Ser-318 ATG13 was from Abnova. Mouse anti α-tubulin was from Merck-Millipore. Mouse anti-LC3 for immunofluorescence was from MBL International, and sheep anti-LC3, used for immunoblotting, was generated by the Division of Signal Transduction Therapy, University of Dundee, from recombinant full-length human GST-LC3b and affinity-purified.

Petherick K.J., Conway O.J., Mpamhanga C., Osborne S.A., Kamal A., Saxty B, & Ganley I.G. (2015). Pharmacological Inhibition of ULK1 Kinase Blocks Mammalian Target of Rapamycin (mTOR)-dependent Autophagy. The Journal of Biological Chemistry, 290(18), 11376-11383.

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Cell Viability Assay with AZD8055

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Cell viability was assessed using the CellTiter-Glo Luminescent Assay (Promega). Cells were plated in triplicates at 1–2 × 10⁴ cells/well in 96-well plate with increase concentrations of AZD8055 (Selleckchem) in 100 μl total volume. Cell viability was assessed at 72 h after treatment.

Pham L.V., Lu G., Tamayo A.T., Chen J., Challagundla P., Jorgensen J.L., Medeiros L.J, & Ford R.J. (2015). Establishment and characterization of a novel MYC/BCL2 “double-hit” diffuse large B cell lymphoma cell line, RC. Journal of Hematology & Oncology, 8, 121.

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Preparation and Characterization of Sodium Alginate Solutions

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Two percent sodium alginate was prepared by dissolving sodium alginate (Sigma-Aldrich) in phosphate-buffered saline (PBS) and sterilizing by heating to 80°C in a water bath for 15 min (Leo et al., 1990 (link)). Anti-CD150-PE, anti-GRPC5C Alexa Fluor-405, and anti-Ki67-Alexa Fluor 405 antibodies for flow cytometry were purchased from R&D Systems. Anti-CD49f-PE and anti-CD90-VioBlue were purchased from Miltenyi Biotec. Transforming growth factor beta-1 (TGFβ-1) and fibroblast growth factor were obtained from PeproTech. Fms-like tyrosine kinase-3 (Flt-3), G-CSF, stem cell factor (SCF), and interferon alpha were purchased from ImmunoTools. All-trans retinoic acid (ATRA) was obtained from Sigma-Aldrich. Hoechst 33342, propidium iodide (PI), and pyronin Y (PY) were purchased from Sigma-Aldrich. The CountBright^TM Absolute Counting Beads was purchased from Thermo Fisher Scientific. MHY1485, AZD8055, rosiglitazone, KU-55933, LEE011, roscovitine (seliciclib, CYC202), glasdegib (PF-04449913), sodium butyrate, dasatinib, BIO, plerixafor (AMD3100), quizartinib, and sorafenib were purchased from Selleckchem. Adiponectin, triglitazone LE135, PD169316, harmine, nilotinib, ethylisopropyl amiloride, anisomycin, and curcumin were purchased from Sigma-Aldrich, while prostaglandin E2 was from BioVision/Cambridge Bioscience. Cytarabine and daunorubicin were purchased from Sigma-Aldrich.

O’Reilly E., Zeinabad H.A., Nolan C., Sefy J., Williams T., Tarunina M., Hernandez D., Choo Y, & Szegezdi E. (2021). Recreating the Bone Marrow Microenvironment to Model Leukemic Stem Cell Quiescence. Frontiers in Cell and Developmental Biology, 9, 662868.

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Chemical Inhibitors of Cell Signaling Pathways

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BEZ235, CAL-101, GSK650394, Roscovitine, Salubrinal, AZD8055, MK-2206 and PF4708671 were from Selleck Chemicals LLC (Houston, TX, USA). CN585 (6-(3,4-Dichlorophenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine) was from Merck Millipore (Merck, Damstadt, Germany). Ionomycin, PMA, CsA, FK-506 and BV-02 were from Sigma-Aldrich.

Tosello V., Saccomani V., Yu J., Bordin F., Amadori A, & Piovan E. (2016). Calcineurin complex isolated from T-cell acute lymphoblastic leukemia (T-ALL) cells identifies new signaling pathways including mTOR/AKT/S6K whose inhibition synergize with calcineurin inhibition to promote T-ALL cell death. Oncotarget, 7(29), 45715-45729.

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