Azd5991

Manufactured by Selleck Chemicals

Sourced in Germany, United States

AZD5991 is a laboratory reagent produced by Selleck Chemicals. It is a small molecule compound used in research applications. The core function of AZD5991 is to serve as a tool compound for scientific investigations.

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Lab products found in correlation

14 protocols using azd5991

Preparation of Iron Chelation Agents

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Deferoxamine (Novartis Pharma SAS) was dissolved in sterile distilled water and deferasirox (Selleckchem S1712), was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 300 and 50 mmol/L, respectively. Ironomycin was a kind gift of Raphaël Rodriguez team (11 (link)), this compound was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mmol/L. Other reagents: erastin (Selleckchem S7242, 10 mmol/L in DMSO), ferrostatin-1 (Selleckchem S7243, 50 mmol/L in DMSO), Q-VD Oph (SelleckChem S7311, 10 mmol/L in DMSO, 30′ pretreatment), iron(III) chloride hexahydrate (31232 Sigma Aldrich, 0.1 mol/L in water, 4 hours after treatment), reduced gluthatione GSH (Sigma Aldrich G4251, 0.1 M in PBS) H₂O₂ (Sigma Aldrich 216763), mafosfamide (surrogate of cyclophosphamide, Santa Cruz ChemCruz SC-211761, 10 mmol/L in saline water), gemcitabine (Sellekchem S1149, 50 mmol/L in saline water), doxorubicin (Sellekchem S1208, 20 mmol/L in DMSO), CldU (Abcam ab213715, 20 mmol/L), IdU (Abcam ab142581, 2 mmol/L), 3-methyl adenine (Merck, #189490), β-mercaptoethanol (Sigma, M3148), staurosporine (Selleckchem, S1421), ketokonazole (Sigma, UC280), Avasimibe (from Sigma, PZ0190), ibrutinib (from Sellekchem S2680), entospletinib (from Selleckchem, S7523), venetoclax (from Sellekchem, S8048), AZD-5991 (from Selleckchem, S8643), and A1155463 (from TargetMol, T6748).

Devin J., Cañeque T., Lin Y.L., Mondoulet L., Veyrune J.L., Abouladze M., Garcia De Paco E., Karmous Gadacha O., Cartron G., Pasero P., Bret C., Rodriguez R, & Moreaux J. (2022). Targeting Cellular Iron Homeostasis with Ironomycin in Diffuse Large B-cell Lymphoma. Cancer Research, 82(6), 998-1012.

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BH3 Profiling of Cell Lines

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BH3 Profiling was performed using the iBH3 plate-based method as previously published (56 (link)). In brief, cell lines were seeded at a density of 1 x 10⁶ cells/mL 24 hours before profiling. Four million cells of each cell line were pelleted at 300 x g for 5 minutes and resuspended in 2mL MEB-P25 (150mM Mannitol, 10mM HEPES-KOH pH 7.5, 150mM KCl, 1mM EGTA, 1mM EDTA, 0.1% BSA, 5 mM Succinate, 0.25% Polaxamer 188[Fisher, MT61161RM]). Cells were permeabilized with digitonin (Sigma, D5628) exposed to BH3 peptides for 60 minutes at 25°C and mitochondrial Cytochrome C release was measured by flow cytometry using a FITC-conjugated antibody (BioLegend, 983502). BH3 peptides were synthesised by New England Peptide using published sequences (100 (link)). The Bcl-XL-selective inhibitor A-1331852 (Selleckchem, S7801), the Bcl-2-selective inihibtor ABT-199 (Selleckchem, S8048) and the MCL-inhibitor AZD5991 (Selleckchem, S8643) were used at a concentration of 10 μM. Results were only deemed valid where cell cytochrome C release in presence of DMSO control was <10% and cytochrome C release in presence of 50ug/mL(25uM) Alamethicin (Enzo, BML-A150-0005) was >90%. Figure represents mean of three independent experiments.

Flümann R., Rehkämper T., Nieper P., Pfeiffer P., Holzem A., Klein S., Bhatia S., Kochanek M., Kisis I., Pelzer B.W., Ahlert H., Hauer J., da Palma Guerreiro A., Ryan J.A., Reimann M., Riabinska A., Wiederstein J., Krüger M., Deckert M., Altmüller J., Klatt A.R., Frenzel L.P., Pasqualucci L., Béguelin W., Melnick A.M., Sander S., Montesinos-Rongen M., Brunn A., Lohneis P., Büttner R., Kashkar H., Borkhardt A., Letai A., Persigehl T., Peifer M., Schmitt C.A., Reinhardt H.C, & Knittel G. (2020). An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities. Blood Cancer Discovery, 2(1), 70-91.

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Cell Death Induction Reagents and Antibodies

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Osimertinib and erlotinib were the same as previously described ^{10 (link)}. Almonertinib was purchased from Advanced ChemBlocks Inc (Burlingame, CA). S63845 (MIK665) and AZD5991 were purchased from Selleckchem (Houston, TX). APG3526 was provided by Ascentage Pharm (Rockville, MD). CYD-2-11 was developed in house by Dr. Xingming Deng’s group as described previously ^{13 , 14 (link)}. Digitonin and bismaleimidohexane (BMH) were purchased from Sigma-Aldrich (St. Louis, MO) and ThermoFischer Scientific (Waltham, MA), respectively. Antibody against Cyt C (sc-13156) was purchased from Santa Cruz Biotechnology (Dallas, Texas). Smac/Diablo antibody (#15108) was purchased from Cell Signaling Technology (Danvers, MA). The remaining antibodies used in this study were described previously ^{10 (link), 25 (link)}.

Ma G., Deng Y., Qian L., Vallega K.A., Zhang G., Deng X., Owonikoko T.K., Ramalingam S.S., Fang D.D., Zhai Y, & Sun S.Y. (2022). Overcoming acquired resistance to third generation EGFR inhibitors by targeting activation of intrinsic apoptotic pathway through Mcl-1 inhibition, Bax activation, or both. Oncogene, 41(12), 1691-1700.

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Mycobacterium tuberculosis Infection Assay

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Single cell suspensions of M.tb in RHH [for MDM infections: 10 mM HEPES (Life Technologies) and 0.1% human serum albumin (CSL Behring, King of Prussia, PA) in RPMI] or DHH [for BMDM infections: 10 mM HEPES and 0.1% human serum albumin in DMEM] were added to the macrophages at MOI 1 and cells were incubated for 2 h at 37 ◦C, with the first 30 min on a platform shaker. Macrophages were then washed and incubated in RPMI with 2% autologous serum (for MDMs) or DMEM with 2% HI-FBS (for BMDMs) for the indicated times. Where indicated, the MCL-1 and BCL-2 inhibitors, antibiotics, or solvent control (dimethyl sulfoxide, DMSO), were added after this wash step. The MCL-1 inhibitors S63845, MIK665, and AZD5991, and BCL-2 inhibitor ABT-199 were purchased from Selleckchem (Houston, TX), and the MCL-1 inhibitor AMG 176 was from MedChemExpress (Monmouth Junction, NJ). The antibiotics RIF and INH were purchased from Sigma (St. Louis, MO). All inhibitors and antibiotics were maintained throughout the course of infection. Cells were observed with an EVOS XL Core Imaging System to ensure monolayer integrity was maintained throughout the course of all experiments (Figs S1 and S3).

Arnett E., Pahari S., Wager C.M., Hernandez E., Bonifacio J.R., Lumbreras M., Renshaw C., Montoya M.J., Opferman J.T, & Schlesinger L.S. (2023). Combination of MCL-1 and BCL-2 inhibitors is a promising approach for a host-directed therapy for tuberculosis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 168, 115738.

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Human HSC Viability and Apoptosis Assays

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For D+Q, ABT-263 or AZD5991 (Selleck) treatment, D37 (5× 10⁴/well, in triplicate), GRO (1× 10⁵/well, in triplicate) or SEN (1.5× 10⁵/well, in quadruplicate) human HSCs were plated in 6-well plates for 24 h, treated with indicated drug combinations/concentrations for 3 (D37) or 5 days (HSCs), trypsinized and counted with a hemocytometer; The viable cell numbers were normalized to those of vehicle groups, and apoptosis was determined by PI/Annexin-V staining and flow cytometry with Calibur (BD Biosciences). For ICM treatment, D37 (5× 10⁴/well, in quadruplicate), GRO (1× 10⁵/well, in triplicate) or SEN (1.5× 10⁵/well, in quadruplicate) human HSCs were plated in 6-well plates for 24 h, treated with indicated concentrations for 72 h, counted and normalizing to vehicle control (ICM: 0 μM). For HMGB1 treatment, human HSCs were seeded in 12-well plates (2×10⁴/well, in triplicate) for 24 h, serum-starved for 6 h, and then cultured in DMEM containing BSA or 1 nM HMGB1 (1690-HMB, R&D) for 15 h.

Li F., Huangyang P., Burrows M., Guo K., Riscal R., Godfrey J., Lee K.E., Lin N., Lee P., Blair I.A., Keith B., Li B, & Simon M.C. (2020). FBP1 loss disrupts liver metabolism and promotes tumourigenesis through a hepatic stellate cell senescence secretome. Nature cell biology, 22(6), 728-739.

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Apoptosis Inducers and Inhibitors Protocol

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APG-1252-M1, APG-1252 (prodrug of APG-1252-M1 for in vivo study) and APG-3526 were provided by Ascentage Pharma Group Inc. (Suzhou, China). ABT263, ABT199 and S63845 (MIK665) were purchased from MedChemExpress (Monmouth Junction). AZD5991 was purchased from Selleckchem (Houston, TX). The caspase inhibitors, CBZ-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) and Z-lle-Glu(Ome)-Thr-Asp(OMe)-fluoromethyl ketone (z-IETD-fmk), were purchased from Enzyme System Products (Livermore, CA). CYD-2-11 was provided by Dr. X. Deng (Emory University, GA). BMH was purchased from Thermo Scientific (Rockford, IL). Rabbit antibodies against caspase-8, caspase-3, PARP, Smac, PUMA and DR5 (D4E9) were purchased from Cell Signaling Technology (Beverly, MA). Mouse antibodies against Bcl-2, Bax (N-20), Mcl-1, cytochrome C (Cyt C) and rabbit anti-Bcl-X_L antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas). Bim antibody was purchased from EMD Millipore (Burlington, MA). Mouse monoclonal DR4 antibody (B-N28) was purchased from Cell Science (Newburyport, MA). Rabbit polyclonal microtubule-associated protein light chain 3 (LC3) antibody was purchased from Novus Biologicals (Littleton, CO). GAPDH antibody was purchased from Trevigen (Gaithersburg, MD). Mouse monoclonal tubulin and actin antibodies were purchased from Sigma Chemical (St. Louis, MO).

Yao W., Bai L., Wang S., Zha Y, & Sun S.Y. (2022). Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL dual inhibitor that induces Bax-dependent apoptosis. Neoplasia (New York, N.Y.), 29, 100798.

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Characterization of Leukemia Cell Lines

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MDS-L cells were a gift from Kaoru Tohyama, MD, PhD (Department of Laboratory Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan), and were cultured as described (1 ). Human myeloblastic leukemia K562, Kasumi1, KG-1 and acute lymphoblastic leukemia CEM were purchased from the Duke Cancer Institute Cell Culture Facility. Cells were thawed and used in experiments within 4 weeks of thawing. Human breast epithelial cells MCF-10A were transduced with either p185 kDa full-length ERBB2 receptor or p110 kDa truncated isoform of ERBB2 under a doxycycline inducible promotor as described (2 (link)). Cell lines were negative for mycoplasma. They were tested for mycoplasma by Mycoplasma PCR Detection kit (Sigma, St. Louis, MO) within 6 months of studies being performed.
Lapatinib was purchased from LC Laboratories (Woburn, MA). Afatinib, neratinib, venetoclax, maritoclax and AZD5991 were purchased from Selleckchem (Houston, TX).

Kam A.Y., Piryani S.O., Lee C.L., Rizzieri D.A., Spector N.L., Sarantopoulos S, & Doan P.L. (2021). Selective ERBB2 and BCL2 Inhibition is Synergistic for Mitochondrial-mediated Apoptosis in MDS and AML cells. Molecular cancer research : MCR, 19(5), 886-899.

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Comprehensive Molecular Profiling Protocol

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Trametinib (GSK1120212) (#S2673), PLX4720 (#S1152), PLX4032 (#S1267), PLX8394 (#S7965), AZD5991 (#S8643), MK2206 (#S1078), buparlisib (BKM120) (#S2247), and Navitoclax (#S1001) were purchased from Selleck Chemicals, LLC. DRP-1 (#8570S), BAK (#12105S), Bcl-XL (2762S), Caspase 9 (#9502S), Cleaved caspase 3 (#9661S), HSP90 (#4877S), pERK1/2 (#9101S), pRb S807/811 (#9308S), pT308 AKT (#2965S), pPRAS40 T246 (#2640S), VCAM1 (#13662S), Mcl-1 (#4572S), LAMP-1 (#9091T), Vinculin (#4650S), Tubulin (#2148S), COX-IV (#11967S), cytochrome c (#11940S) cleaved PARP (#9541) and GAPDH (#2118S) antibodies were purchased from Cell Signaling Technology. ERK2 (sc-1647) and AIF (#sc-13116) were purchased from Santa Cruz Biotechnology Inc., γH2AX (ab11174) was purchased from Abcam, Actin (A2066) was purchased from Sigma-Aldrich Co., and Bcl-2 (#51-6511GR) was purchased from BD Biosciences. Goat anti-rabbit IgG Alexa Fluor^TM 488 secondary antibody was purchased from Invitrogen.

Cavallo M.R., Yo J.C., Gallant K.C., Cunanan C.J., Amirfallah A., Daniali M., Sanders A.B., Aplin A.E., Pribitkin E.A, & Hartsough E.J. (2024). Mcl-1 mediates intrinsic resistance to RAF inhibitors in mutant BRAF papillary thyroid carcinoma. Cell Death Discovery, 10, 175.

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Preparation of BH3 Mimetic Compounds

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The BH3 mimetic compounds ABT-263, ABT-199, ABT-737, AZD5991, Gambogic acid, Gossypol, Obatoclax, Sabutoclax, S55746, and TW-37 (Selleckchem, Cologne, Germany) were dissolved in DMSO to obtain 10 mM stock solutions.

Castillo Bautista C.M., Eismann K., Gentzel M., Pelucchi S., Mertens J., Walters H.E., Yun M.H, & Sterneckert J. (2023). Obatoclax Rescues FUS-ALS Phenotypes in iPSC-Derived Neurons by Inducing Autophagy. Cells, 12(18), 2247.

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Apoptosis Signaling Pathway Inhibitors

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ABT-263, AZD5991, Mitoxantrone, PF-543, Thapsigargin, KIRA6, GSK2606414, Necrostatin-1, Disulfiram and z-IETD-fmk were all purchased from Selleckchem (Houston, TX, USA). Fumonisin B1 was from Cayman Chemicals (Ann Arbor, MI, USA). All compounds were prepared in a DMSO vehicle. Anti-Calreticulin, anti-phospho eIF2α Ser51, anti-full length Caspase 8, anti-Bap31, anti-Cleaved PARP, anti-GRP78/BiP, anti-Bcl-XL, anti-FLIP, anti-full length BID, and anti-total SphK1 antibodies were from Cell Signaling Technologies (Beverly, MA, USA). Anti-GAPDH, and anti-CerS6 antibodies were from Santa Cruz Biotechnologies (Dallas, TX, USA). Anti-phospho-SphK1 Ser225 antibodies were from ECM Biosciences (Versailles, KY, USA). Anti-CerS5 antibodies were from LSBio (Seattle, WA, USA).

Hengst J.A., Nduwumwami A.J, & Yun J.K. (2022). Regulatory Role of Sphingosine-1-Phosphate and C16:0 Ceramide, in Immunogenic Cell Death of Colon Cancer Cells Induced by Bak/Bax-Activation. Cancers, 14(21), 5182.

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