Reversine

Manufactured by Merck Group

Sourced in United States

Reversine is a small molecule compound used in cell biology research. It functions as an inhibitor of the enzyme BuBR1, which plays a role in mitotic spindle assembly and chromosome segregation. Reversine can be used to study cellular processes related to cell division and chromosome dynamics.

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Lab products found in correlation

Nocodazole, by Merck Group (12 mentions) Mg132, by Merck Group (10 mentions) Monastrol, by Merck Group (4 mentions) Thymidine, by Merck Group (4 mentions) Gsk923295, by Selleck Chemicals (3 mentions) Bi2536, by Selleck Chemicals (3 mentions) Roscovitine, by Merck Group (3 mentions) Paclitaxel, by Merck Group (3 mentions) Doxycycline, by Merck Group (3 mentions) Ro 3306, by Merck Group (3 mentions) Protease inhibitors cocktail, by Merck Group (2 mentions) Vx 680, by Selleck Chemicals (2 mentions) Nu9056, by Bio-Techne (2 mentions) Zm447439, by Bio-Techne (2 mentions) Nms p715, by Merck Group (2 mentions) Zoledronic acid, by Merck Group (2 mentions) Aspirin, by Merck Group (2 mentions) Rosiglitazone, by Merck Group (2 mentions) Resveratrol, by Merck Group (2 mentions) Indomethacin, by Merck Group (2 mentions)

33 protocols using reversine

Isolation and Culture of Cerebellar Granule Neurons

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CGNPs for cell culture and cell sorting were harvested from transgenic cerebella, as described previously [18 (link)]. In short, cerebella were dissected and meninges were removed. A single cell suspension was prepared with a papain dissociation kit, according to the manufacturer’s instructions (Worthington, Lakewood, NJ, USA).
For cell culturing, unsorted P0 and P7 CGNPs were resuspended in culture media (DMEM-F12 (Gibco, New York, NY, USA) supplemented with 1% N2 (Invitrogen, Waltham, MS, USA); 1.5% glucose (Invitrogen); 5 μM HEPES (Invitrogen); and 0.25 μg/mL SHH (R&D Systems, Minneapolis, MN, USA)). The cells were seeded on pre-coated plates with poly-D-lysine (100 μg/mL, Sigma) at a density of 250.000 cells per well in 24-well plates. For ionizing radiation experiments, the cells were irradiated the next morning with 0.5, 1 or 2 Gray of Y-rays from a ¹³⁷Cesium source (IBL 637 Cesium-137 Y-ray-machine). The cells were fixed at 2 h post irradiation. For reversine treatment, the cells were treated with DMSO or 250 nM of reversine (Sigma) for 48 h and subsequently fixed.
For gDNA, RNA, and protein isolations, tdTomato positive/DAPI negative cells were sorted on a Beckman Coulter MoFlo Astrios sorter (Brea, CA, USA). Cell pellets were stored at −80 °C until further processing. For the isolation of nuclei for scWGS, see below.

Bočkaj I., Martini T.E., Smit M.J., Armandari I., Bakker B., Wardenaar R., Meeuwsen-de Boer T.G., Bakker P.L., Spierings D.C., Hoving E.W., Guryev V., Foijer F, & Bruggeman S.W. (2022). Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum. International Journal of Molecular Sciences, 23(17), 9852.

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High-Throughput Screening of siRNA Impacts

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The primary screen was performed using the human siRNA Smart-pool siGenome library from Dharmacon, whereas the secondary screen was performed using the corresponding deconvoluted siRNA duplexes (where four siRNAs included in the pool were tested independently). Contact-inhibited RPE1 cells were reverse-transfected in triplicate 384-well plates using Lullaby transfection reagent [OZ Biosciences, 800 cells/well, 0.2 μL Lullaby/well, 18.75 nmol/L siRNA, 250 nmol/L reversine (Sigma)] using a Biomek FX liquid handling robot (Beckman). Cells were cultured for 96 hours and fixed with 4% formaldehyde (ThermoFisher, no. 28908), except for the deconvolution experiment, where cells were pulsed for 1 hour with 10 μmol/L EdU before fixation to label cells in S phase. Cells were subsequently permeabilized and blocked in PBS + 3% BSA + 0.2% Triton-X100 for 30 minutes and processed for immunofluorescence (IF) and EdU detection (LifeTechnology) according to the manufacturer’s protocol. Images were acquired and analyzed using an ArrayScan VTi-automated microscope (Cellomics), except for total cell counts, which were acquired on an Acumen Explorer eX3 laser scanning microplate cytometer (TTPLabtech). For each parameter, a median Z-score was derived using plate normalization [(well value – plate median)/plate Median Absolute Deviation].

Sansregret L., Patterson J.O., Dewhurst S., López-García C., Koch A., McGranahan N., Chao W.C., Barry D.J., Rowan A., Instrell R., Horswell S., Way M., Howell M., Singleton M.R., Medema R.H., Nurse P., Petronczki M, & Swanton C. (2017). APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability. Cancer discovery, 7(2), 218-233.

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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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Microscopy Imaging Methodology with Inhibitors

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Inhibitors were dissolved in DMSO and were used at the following concentrations: 50 nM CENP-E inhibitor (GSK923295, Cambridge Bioscience), 2–3 μM MPS1 inhibitor (NMS-P715, Merck Chemicals) and 0.15–0.45 μM reversine (Sigma-Aldrich).
Antibodies were used as follows: mouse α-tubulin (Thermo Fisher Scientific, 32-2500), 1:5000 (western blotting or WB) and 1:500 (immunofluorescence or IF); rabbit α-tubulin (Abcam, ab52866), 1:500 (IF); mouse CENP-A (Abcam, ab13939), 1:200 (IF); human CREST (Antibodies Incorporated, 15-234-0001), 1:300 (IF); rabbit pericentrin (Abcam, ab4448), 1:2000 (IF); rabbit KIF11 (Novus Biologicals, NB5000-181), 1:1000 (IF); mouse p53 (Cell Signaling Technologies, 48818S), 1:1000 (IF); and rabbit MAD2 (Millipore UK, MABE866), 1:1000 (WB) and 1:500 (IF).

Dale K.L., Armond J.W., Hynds R.E, & Vladimirou E. (2022). Modest increase of KIF11 expression exposes fragilities in the mitotic spindle, causing chromosomal instability. Journal of Cell Science, 135(17), jcs260031.

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Reversine Cytotoxicity Evaluation in Cells

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Reversine (CAS NO. 656820-32-5) was purchased from Sigma-Aldrich (USA), which was dissolved in dimethyl sulfoxide (DMSO) (Sigma, USA) in accordance with the reagent instruction. Exactly 5000 cells were plated onto 96-well tissue-culture plates and grown in the above-mentioned medium. After overnight attachment, the cells were treated with medium only (containing 0.01% DMSO) as control or medium containing different concentrations of Reversine. After incubation for 24 or 48 h, the number of metabolically active cells was determined using Cell Counting Kit-8 (CCK-8) assay (Dojindo, Shanghai, China). CCK-8 labeling reagent was added to the fresh medium, and the cell were incubated for 1 h at room temperature. Optical density value was examined at 520 nm by using a microplate reader (BioTek, USA). Results were analyzed through statistical methods in three independent studies. The percentage of cell viability was calculated relative to the control wells designated as 100% viable cells.

Huang D., Huang Y., Huang Z., Weng J., Zhang S, & Gu W. (2019). Relation of AURKB over-expression to low survival rate in BCRA and reversine-modulated aurora B kinase in breast cancer cell lines. Cancer Cell International, 19, 166.

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Concentration-Dependent Drug Effects

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Aspirin, indomethacin, resveratrol, reversine, rosiglitazone, and zoledronic acid were purchased from Sigma-Aldrich. Bortezomib was from LC Laboratories. Pamidronate and diphenhydramine were from Massachusetts General Hospital (MGH) Pharmacy. Duration of drug treatment in vitro was 48 hours for all drugs. Concentrations used for drug treatment on the mono-culture and co-cultures are based on the half maximal effective concentrations (EC₅₀) of the selected drugs: Aspirin: 10 μM (for COX-2 inhibition); Bortezomib: 20 nM; diphenhydramine: 10 μM; indomethacin: 40 μM; pamidronate: 20 μM; resveratrol: 20 μM; reversine: 5 μM; rosiglitazone: 100 nM; zoledronic acid: 40 μM.

Shen K., Luk S., Hicks D.F., Elman J.S., Bohr S., Iwamoto Y., Murray R., Pena K., Wang F., Seker E., Weissleder R., Yarmush M.L., Toner M., Sgroi D, & Parekkadan B. (2014). Resolving Cancer-Stroma Interfacial Signaling and Interventions with Micropatterned Tumor-Stromal Assays. Nature communications, 5, 5662.

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Synchronizing and Arresting Cell Lines

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All cell lines were maintained at 37°C with 5% CO₂ (see Supplemental Experimental Procedures for details of origin and media). hTERT-RPE-1 H2B-RFP stable cell lines were generated after transfection with lentiviral construct H2B-RFP (26001; Addgene). RNAi was achieved by transfection of cells for 48 hr with 30 nM small interfering RNA (siControl [D-001210-02] and siWAPL SMART pool [M-026287-01]; Dharmacon) using Lipofectamine RNAiMAX (Invitrogen) and Optimem (Gibco). Drug concentrations used were 10 μM MG132, 100 ng/mL nocodazole, 10 μM S-trityl-L-cysteine (STLC), 100 μM monastrol, and 250 nM reversine (all from Sigma-Aldrich). Release from mitotic arrest was achieved by washing drug out of cells with prewarmed media three to five times, then leaving in incubator for 1 hr (nocodazole), 1.5 hr (STLC and monastrol), or 2.5 hr (MG132).

Worrall J.T., Tamura N., Mazzagatti A., Shaikh N., van Lingen T., Bakker B., Spierings D.C., Vladimirou E., Foijer F, & McClelland S.E. (2018). Non-random Mis-segregation of Human Chromosomes. Cell Reports, 23(11), 3366-3380.

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Depletion and Mutagenesis of Mitotic Regulators

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Drug treatments were realized as follows: nocodazole (Sigma, as indicated, 16h), thymidine (Acros Organics, 2 mM for 16 h), reversine (Sigma, 0.5 μM, 30min), taxol (Calbiochem, 0.2 μM, 16h). For experiments with stable cell lines, 24h before harvesting cells, 1μg/ml of tetracycline was added to express the protein of interest. Plasmid transfections were performed with PEI (polyethylenimine) at a 15:2 (PEI/DNA) ratio for HEK293T cells and with TransIT® (Mirus Bio) for HeLa S3 cells according to the manufacturer’s instructions. Endogenous protein depletion was carried out with DsiRNAs (IDT), using Oligofectamine(Invitrogen) for HEK293T cells and HeLa-S3 or JetPRIME® (Polyplus-transfection) for stable cell line HeLa Flp-In Trex and analyzed at 48-72h after transfection. The Mps1 and Hec1 DsiRNA targets the following sequence:

Mps1 A: 5′-GUAGAUUUCCACAGGGAUUCAAGAGUA- 3′

Mps1 B: 5′-AUACAGUGCCAUAAGUGGUUGCUAUUU-3′

Site-directed mutagenesis was performed using PCR and overlapping mutagenic primers. All constructs were verified by sequencing.

Combes G., Barysz H., Garand C., Gama Braga L., Alharbi I., Thebault P., Murakami L., Bryne D.P., Stankovic S., Eyers P.A., Bolanos-Garcia V.M., Earnshaw W.C., Maciejowski J., Jallepalli P.V, & Elowe S. (2018). Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint. Current Biology, 28(6), 872-883.e5.

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Conditional Aurora-A Knockout and Cell Line Protocols

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Aurora-A conditional knockout mice and MEF cells were generated as previously described [17 (link)]. Aurora-A depletion was induced by 4-OHT according to the protocol described previously [17 (link)]. MEF, RPE1, HeLa, HEK293T and Ptk2 cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and penicillin/streptomycin (100 IU ml⁻¹ and 100 mg ml⁻¹, respectively; Beyotime Biotechnology, Jiangsu, China). Cells were maintained at 37 °C in a 5% CO₂ atmosphere and seeded onto coverslips 24–48 h before experimentation. Transfections were performed with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). MLN8237 (50 nM for human cell lines and 100 nM for Ptk2 cells), Hesperadin (100 nM), BI2536 (100 nM), Reversine (500 nM) and Noc (0.5 μM for human cell lines and 3.3 μM for MEF and 10 μM for Ptk2 cells) were purchased from Sigma (St Louis, MO, USA) or Selleck Chemicals (LLC, Houston, TX, USA), and resolved in DMSO.

Yu F., Jiang Y., Lu L., Cao M., Qiao Y., Liu X., Liu D., Van Dyke T., Wang F., Yao X., Guo J, & Yang Z. (2017). Aurora-A promotes the establishment of spindle assembly checkpoint by priming the Haspin-Aurora-B feedback loop in late G2 phase. Cell Discovery, 3, 16049-.

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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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