Gsk923295

Manufactured by Selleck Chemicals

GSK923295 is a small molecule compound that functions as a selective inhibitor of the Aurora B kinase enzyme. It is a laboratory reagent intended for research use only.

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Lab products found in correlation

Nocodazole, by Merck Group (7 mentions) Mg132, by Merck Group (3 mentions) Monastrol, by Merck Group (3 mentions) Reversine, by Merck Group (3 mentions) Bi2536, by Selleck Chemicals (3 mentions) Doxycycline hyclate, by Merck Group (3 mentions) S trityl l cysteine stlc, by Merck Group (3 mentions) Protame apc ci, by R&D Systems (3 mentions) Az 3146 mps1i, by Selleck Chemicals (3 mentions) Ispinesib, by Selleck Chemicals (2 mentions) Protease inhibitors cocktail, by Merck Group (2 mentions) Roscovitine, by Merck Group (2 mentions) Vx 680, by Selleck Chemicals (2 mentions) Nu9056, by Bio-Techne (2 mentions) Zm447439, by Bio-Techne (2 mentions) Taxol, by Merck Group (2 mentions) Tubastatin a, by Selleck Chemicals (2 mentions) Taxol, by Selleck Chemicals (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) Mg 132 mg132, by Enzo Life Sciences (2 mentions)

13 protocols using gsk923295

Mitotic Spindle Inhibitor Efficacy

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Drugs, suppliers, and concentrations used were Barasertib (Aurora B inhibitor; alternative name AZD1152‐HQPS; SelleckChem S1147; 1.11 nM); CHR‐6494 (Haspin inhibitor; MedChem Express HY‐15217; 500 nM); CW069 (HSET inhibitor; SelleckChem S7336; 25.0 μM); Etoposide (Topoisomerase II inhibitor; SelleckChem S1225; 333 nM); GSK461364 (PLK1 inhibitor; SelleckChem S2193; 2.20 nM); GSK923295 (CENP‐E inhibitor; SelleckChem S7090; 3.20 nM); Ispinesib (KIF11 inhibitor; alternative name SB‐715992; SelleckChem S1452; 1.70 nM); MK‐5108 (Aurora A inhibitor; alternative name VX‐689; SelleckChem S2770; 0.576 nM); MK‐8776 (CHK1 inhibitor; alternative name SCH 900776; SelleckChem S2735; 9.00 nM); Paclitaxel (microtubule inhibitor; SelleckChem S1150; 2.67 nM); Vinblastine (microtubule inhibitor; Sigma V1377; 2.40 nM); and YM155 (BIRC5 inhibitor; SelleckChem S1130; 0.540 nM).

Maier L.J., Kallenberger S.M., Jechow K., Waschow M., Eils R, & Conrad C. (2018). Unraveling mitotic protein networks by 3D multiplexed epitope drug screening. Molecular Systems Biology, 14(8), e8238.

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Mitotic Inhibitors and Modifiers Protocol

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Nocodazole (100 ng/mL, ≥99%), monastrol (50 μM, ≥98%), MG132 (10 μM, ≥90%), OA (500 nM, ≥92%), Reversine (1 μM, ≥98%), Roscovitine (20 μM, ≥98%), NAM (5 mM, ≥99.5%), and TSA (1 μM, ≥98%) were from Sigma. MG149 (100 μM, >99%) was from Axon. NU9056 (20 μM, >98%), ZM447439 (2 μM, >99%) were from Tocris Bioscience. GSK923295 (50 nM, >99%), BI2536 (100 nM, >99%), VX-680 (500 nM, >99%) was from Selleckchem. The protease inhibitors cocktail was from Sigma.

Mo F., Zhuang X., Liu X., Yao P.Y., Qin B., Su Z., Zang J., Wang Z., Zhang J., Dou Z., Tian C., Teng M., Niu L., Hill D.L., Fang G., Ding X., Fu C, & Yao X. (2016). Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation. Nature chemical biology, 12(4), 226-232.

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Taxol Cytotoxicity Experiments Optimization

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For the taxol cytotoxicity experiments in Fig. 3C, C’, taxol (Sigma-Aldrich, T7191) was serially diluted (24; 12; 6; 3; 1.5; 0.75; 0.375 nM) and the different concentrations added for 120 h. The same concentrations of taxol were added in all other cytotoxicity experiments for 72 h. Pharmacological inhibition of HDAC6, and increasing of α-tubulin acetylation was obtained using 1.5 μM of Tubastatin A (Selleck Chemicals, S8049) for 24 h. 20 nM of GSK923295 (CENP-E inhibitor) (Selleck Chemicals, S7090) was added along with taxol in Fig. 6G-I. 5 μM of MG132 (Calbiochem, 474790) was used to induce metaphase arrest (less than 2 h of treatment to avoid cohesion fatigue). For live-cell and fixed cell analysis, 6 nM of taxol was added before acquisition or for 24 h before fixation, respectively. DMSO was used as control for these drugs.

Lopes D., Seabra A.L., Orr B, & Maiato H. (2022). α-Tubulin detyrosination links the suppression of MCAK activity with taxol cytotoxicity. The Journal of Cell Biology, 222(2), e202205092.

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Taxol Cytotoxicity Assays and Inhibitors

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For the taxol cytotoxicity experiments in Fig. 3, C and C′, taxol (T7191; Sigma-Aldrich) was serially diluted (24; 12; 6; 3; 1.5; 0.75; 0.375 nM), and the different concentrations added for 120 h. The same concentrations of taxol were added in all other cytotoxicity experiments for 72 h. Pharmacological inhibition of HDAC6 and increasing α-tubulin acetylation was obtained using 1.5 µM of Tubastatin A (S8049; Selleck Chemicals) for 24 h. 20 nM of GSK923295 (CENP-E inhibitor; S7090; Selleck Chemicals) was added along with taxol in Fig. 6, G−I. 5 µM of MG132 (474790 Calbiochem) was used to induce metaphase arrest (less than 2 h of treatment to avoid cohesion fatigue). For live- and fixed-cell analysis, 6 nM of taxol was added before acquisition or for 24 h before fixation, respectively. DMSO was used as control for these drugs.

Lopes D., Seabra A.L., Orr B, & Maiato H. (2022). α-Tubulin detyrosination links the suppression of MCAK activity with taxol cytotoxicity. The Journal of Cell Biology, 222(2), e202205092.

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Chromosome Alignment and Lagging Assay in HeLa Cells

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HeLa S3 cells were cultured in DMEM (Hyclone) supplemented with 10% (vol/vol) of bovine growth serum and Pen/Strep (100 μg/ml, Hyclone) at 37°C and 5% CO₂. For measuring chromosome alignment, cells seeded onto coverslips were arrested with STLC (Sigma, 5 μM) for 12 h. Subsequently, cells were released into MG132 (Calbiochem, 20 μM) for 2.5 h and then fixed with PTEMF (0.2% Triton X-100, 20 mM PIPES, pH 6.9, 1 mM MgCl₂, 10 mM EGTA, and 4% formaldehyde). Cells were also treated with either Aurora B (ZM-447439, Enzo) or CENP-E (GSK923295, Selleckchem, S7090) inhibitors at the indicated concentrations for 30 min, before being fixed and stained. To measure lagging chromosomes in anaphase, cells were arrested for 16 h in G2/M with 4 µM RO-3306 before being released for 1.5–2 h into control media or media containing ZM447439. Primary antibodies for immunofluorescence were used at 1 µg/ml and included CREST anti-Centromere serum (HCT-0100, Immunovision), anti-α-Tubulin (DM1A, Santa Cruz), and anti-γ-Tubulin (T3559, Sigma-Aldrich). Hoechst 33342 (Thermo Scientific) was used at 1 mg/ml.

Gama Braga L., Prifti D.K., Garand C., Saini P.K, & Elowe S. (2021). A quantitative and semiautomated method for determining misaligned and lagging chromosomes during mitosis. Molecular Biology of the Cell, 32(9), 880-891.

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Immunofluorescence and Western Blot Assays

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Anti-α-tubulin antibody (mouse, FITC-DM1A, F2168; Sigma-Aldrich), anti-CENP-A antibody (mouse, ab13939; Abcam), anti-MKLP1 antibody (rabbit, ab204478; Abcam), and Rhodamine-conjugated phalloidin (R415; Invitrogen) were used for immunofluorescence. The appropriate secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. Antibodies used for western blots were anti-α-tubulin (mouse, DM1A, T9017; Sigma-Aldrich) and anti-BubR1 (mouse, 612503; BD Biosciences). Anti-CENP-E antibody (HpX) was generated as previously described (Yao et al., 1997 (link)). Anti-PRC1 antibody used in immunofluorescence and western blot was generated as described (Fu et al., 2007 (link)).
Nocodazole (100 ng/ml), monastrol (50 μM), MG132 (10 μM), and reversine (300 nM) were from Sigma. GSK923295 (100 nM) and BI2536 (100 nM) were from Selleckchem. Syntelin (1 μM) was synthesized as described before (Ding et al., 2010 (link)). The protease inhibitor cocktail was from Sigma.

Liu X., Xu L., Li J., Yao P.Y., Wang W., Ismail H., Wang H., Liao B., Yang Z., Ward T., Ruan K., Zhang J., Wu Q., He P., Ding X., Wang D., Fu C., Dou Z., Yan F., Wang W., Liu X, & Yao X. (2019). Mitotic motor CENP-E cooperates with PRC1 in temporal control of central spindle assembly. Journal of Molecular Cell Biology, 12(8), 654-665.

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Mitotic Inhibitors and Modifiers Protocol

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Synthesis and Procurement of Diverse Compounds

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KIF18A compounds (AM-0277, AM-1882, AM-5308, AM-9022) were synthesized by Amgen. The following compounds were purchased as follows: AM-7710 (Enamine), BI-2536 (Jubilant Biosys), docetaxel (Accord Healthcare), doxorubicin (Sigma-Aldrich), gemcitabine (Zydus Hospira), GF120918 (Sigma-Aldrich), GSK923295 (Selleck), ispinesib (Selleck), nocodazole (Sigma-Aldrich), nutlin 3a (Cayman Chemical), olaparib (AstaTech), paclitaxel (Sigma-Aldrich), palbociclib (Sigma-Aldrich) and vincristine (Tocris).

Payton M., Belmontes B., Hanestad K., Moriguchi J., Chen K., McCarter J.D., Chung G., Ninniri M.S., Sun J., Manoukian R., Chambers S., Ho S.M., Kurzeja R.J., Edson K.Z., Dahal U.P., Wu T., Wannberg S., Beltran P.J., Canon J., Boghossian A.S., Rees M.G., Ronan M.M., Roth J.A., Minocherhomji S., Bourbeau M.P., Allen J.R., Coxon A., Tamayo N.A, & Hughes P.E. (2023). Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers. Nature Cancer, 5(1), 66-84.

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Cell Culture and Drug Treatment Protocol

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HeLa, hTERT-RPE1, U2OS, A549, DLD-1, and NIH/3T3 cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin, and 2 mM L-glutamine at 37°C with 5% CO₂. The HEC-6 cell line was cultured in DMEM supplemented with 15% FBS and 2 mM L-glutamine. Doxycycline-inducible cell lines were cultured in medium containing FBS certified tetracycline-free. spCas9 expression in inducible CRISPR/Cas9 cell lines was induced with 1 μg/ml doxycycline hyclate (Sigma) at 24 hr intervals for 2 days. All other doxycycline-inducible constructs were induced with 10 ng/ml doxycycline hyclate, unless indicated in figure legend. Other drugs used on human cells were Nocodazole (Sigma, 330 nM), S-trityl-L-cysteine (STLC; Sigma, 10 μM unless otherwise indicated), Paclitaxel (Taxol; Invitrogen, 1 μM), GSK923295 (CENP-E inhibitor; Selleck Chemicals, 100 nM), proTAME (APC/Ci; R&D Systems, 12 μM), AZ-3146 (Mps1i; Selleck Chemicals, 4 μM), cycloheximide (CHX; Sigma, 50 μg/ml), MG-132 (MG132; Enzo Life Sciences, 10 μM) unless concentration indicated otherwise in figure legend. Cells were enriched in mitosis with treatment with 330 nM Nocodazole for 16-17 hrs or if indicated, 10 μM STLC for 18 hrs. The antibodies used in this study are described in the relevant methods and in Supplementary Table 1.

Dikalova A.E., Kadiiska M.B, & Mason R.P. (2001). An in vivo ESR spin-trapping study: Free radical generation in rats from formate intoxication— role of the Fenton reaction. Proceedings of the National Academy of Sciences of the United States of America, 98(24), 13549-13553.

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Reversine, HDAC, and EZH2 Inhibitor Treatments

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Cells were treated with reversine (Cayman Chemical Company) at a concentration of 0.5 µM for either 24 h (for MDA-MB-231) or 48 h (4T1). The HDAC inhibitor vorinostat (Sigma-Aldrich; also known as SAHA) was used at a concentration of 0.5 µM for 24 h, and the methyltransferase inhibitor GSK-126 (XcessBio; EZH2) was used at a concentration of 5 µM for 5 days. Long-term reversine treatment was done 24 h after cells were passaged, and the medium was replaced with normal cell medium after 48 h. GSK923295 (Selleck Chemicals) was used at the concentration of 50 nM for 24 h. DMSO was used as the vehicle control.

Agustinus A.S., Al-Rawi D., Dameracharla B., Raviram R., Jones B.S., Stransky S., Scipioni L., Luebeck J., Di Bona M., Norkunaite D., Myers R.M., Duran M., Choi S., Weigelt B., Yomtoubian S., McPherson A., Toufektchan E., Keuper K., Mischel P.S., Mittal V., Shah S.P., Maciejowski J., Storchova Z., Gratton E., Ly P., Landau D., Bakhoum M.F., Koche R.P., Sidoli S., Bafna V., David Y, & Bakhoum S.F. (2023). Epigenetic dysregulation from chromosomal transit in micronuclei. Nature, 619(7968), 176-183.

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