AZ3146 (2 µM, Tocris) and proTAME (Boston Chemicals) were added just prior to filming at indicated concentrations. Cells were treated with 25 ng/ml nocodazole (Sigma Aldrich) overnight to arrest them. For SAC silencing assays (Fig. S1A ) 12.5 ng/ml nocodazole was used in the media and reversine (Cayman Chemicals) added after 60 min of filming. Cyclin B1 degradation assays were performed similarly but with 25 ng/ml nocodazole.
Reversine
Manufactured by Cayman Chemical
Sourced in United States
Reversine is a laboratory product available from Cayman Chemical. It functions as a small molecule inhibitor. Reversine is intended for research use only and has not been approved for human or veterinary use.
Automatically generated - may contain errors
Lab products found in correlation
Nocodazole, by Merck Group (8 mentions)
Thymidine, by Merck Group (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Az3146, by Bio-Techne (2 mentions)
Bafa1, by Merck Group (2 mentions)
Ro 3306, by Merck Group (2 mentions)
Zm447439, by Bio-Techne (2 mentions)
Nocodazole, by Cayman Chemical (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Hygromycin b, by Santa Cruz Biotechnology (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Rpe 1 cells, by American Type Culture Collection (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Str profiling, by Eurofins (1 mentions)
C2211, by Merck Group (1 mentions)
Gw843682x, by Bio-Techne (1 mentions)
Bjhtert, by American Type Culture Collection (1 mentions)
Imr 90, by American Type Culture Collection (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Staurosporine, by Merck Group (1 mentions)
23 protocols using reversine
1
Cell Cycle Arrest and Silencing Assays
2
Cell Line Maintenance and Treatment
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RPE1 cells were purchased from ATCC, and HeLa Flp‐in cells were a gift from S Taylor (University of Manchester, UK) (Tighe et al, 2008 ). The RPE1 Cyclin B1‐EYFP cells have been published previously (Shaltiel et al, 2014 ), as have the U2OS with LacO array on chromosome 1 (Janicki et al, 2004 ). All cells were authenticated by STR profiling (Eurofins) and screened every 4–8 weeks to ensure they were mycoplasma‐free. Cells were cultured in DMEM supplemented with 9% FBS and 50 μg/ml penicillin/streptomycin, except during fluorescence time‐lapse analysis, when they were cultured in Leibovitz's L‐15 media (900 mg/l D+ Galactose, 5 mM Sodium Pyruvate, no phenol red). Doxycycline (1 μg/ml), STLC (S‐Trityl‐l ‐cysteine: 10 μM) and thymidine (2 mM) were purchased from Sigma‐Aldrich, nocodazole (3.3 μM) from Millipore, puromycin and hygromycin B from Santa Cruz Biotechnology, MG132 (10 μM) from SelleckBio, AZ‐3146 (at indicated concentrations) from Axon, rapamycin (100 nM) from LC Laboratories and reversine (at indicated concentrations) from Cayman Chemicals.
3
Mitotic Spindle Inhibitors and Cell Cycle Modulators
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Nocodazole (M1404; Sigma Aldrich) was used at 3.3 μM, MG132 at 10 μm (C2211, Sigma Aldrich), GW843682X at 1 μm (2977, Tocris, Bristol, UK), BI 6727 at 100 nM (S2235, Stratech Scientific, Suffolk, UK), Hesperadin at 100 nM or 500 nM (2096, Axon Medchem, Groningen, The Netherlands), Reversine at 100 nM or 500 nM (10004412, Cayman Chemical, Michigan, USA), 5-Iodotubercidin at 10 μm (HY-15424, Axon Medchem). DMSO was used as a control in the experiments.
4
Culturing and Treating Cell Lines
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Cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Cells were grown at 37°C with 5% CO2 in a humidified environment. For experiments involving drug treatments, controls represent cells treated with vehicle alone. RPE1-hTERT cells were kindly provided by Iain Cheeseman. RPE1-hTERT p53 CRISPR cells were kindly provided by Prasad Jallepalli through David Pellman. The lymphoblastoid cell line used in Supplemental Figure 3C has been previously characterized (Fry et al. 2008 (link)) and was kindly provided by Leona Samson through Mike Hemann. The BJ-hTERT and IMR-90 cell lines were obtained from American Type Culture Collection.
Reversine was obtained from Cayman Chemical, and AZ3146 was obtained from Tocris. BafA1, chloroquine, ammonium chloride, 3-MA, staurosporine, and thymidine were obtained from Sigma-Aldrich. MG132 was purchased from EMD Biosciences.
Reversine was obtained from Cayman Chemical, and AZ3146 was obtained from Tocris. BafA1, chloroquine, ammonium chloride, 3-MA, staurosporine, and thymidine were obtained from Sigma-Aldrich. MG132 was purchased from EMD Biosciences.
5
Oocyte Isolation and Manipulation Protocol
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For oocyte isolation 8–12-week-old CD1, BDF1, FVB or H2B-EGFP [69 (link)] mice were stimulated by 5 IU of pregnant mare's serum gonadotropin (PMSG) 46 hours before experiment and killed by cervical dislocation. Oocytes were collected into the M2 medium and cultured either in Opti-MEM medium (Life Technologies) supplemented with 10% FCS in 5% CO2 atmosphere or in M2 at 37°C. Meiotic maturation was prevented by 2.5 μM milrinone (Sigma-Aldrich). BI2536 (ChemieTek or Axon Medchem BV), reversine (Cayman Chemical or Merck Millipore), MG132 (Calbiochem or Sigma-Aldrich) and flavopiridol (Sigma Aldrich) were used at 100 nM, 1 μM, 1 μM, and 1 μM respectively.
We microinjected oocytes[70 (link)] with 2 pl mEGFP-PLK1, 1 pl EGFP-CENP-C[31 (link)], 4pl 3mCherry-CENP-C[31 (link)], 0.2 pl H2B-mCherry[31 (link)], 1.5 pl EGFP-MAP4[29 (link)], 1 pl securin-EGFP[71 (link)], 0.5 pl cyclin B1-EGFP[72 (link)], and 1 pl mEGFP-EMI1, from 1 μg/μl mRNA stocks. For nuclear envelope permeability measurement, H2B-EGFP oocytes were microinjected with 5 pl of 2 mg/ml 70-kDa dextran conjugated with tetramethylrhodamine (TAMRA) (Sigma Aldrich, D-1819).
We microinjected oocytes[70 (link)] with 2 pl mEGFP-PLK1, 1 pl EGFP-CENP-C[31 (link)], 4pl 3mCherry-CENP-C[31 (link)], 0.2 pl H2B-mCherry[31 (link)], 1.5 pl EGFP-MAP4[29 (link)], 1 pl securin-EGFP[71 (link)], 0.5 pl cyclin B1-EGFP[72 (link)], and 1 pl mEGFP-EMI1, from 1 μg/μl mRNA stocks. For nuclear envelope permeability measurement, H2B-EGFP oocytes were microinjected with 5 pl of 2 mg/ml 70-kDa dextran conjugated with tetramethylrhodamine (TAMRA) (Sigma Aldrich, D-1819).
6
Gastric Cancer Cell Line Culture
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The two human GC cell lines AGS and NCI-N87, in addition to the human immortalized gastric epithelial cell line (GES-1) were obtained from the American Type Culture Collection. GC cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and penicillin-streptomycin (100 U/ml) at 37˚C with 5% CO2. By contrast, GES-1 cells were grown in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS with 100 U/ml P/S at 37˚C with 5% CO2. Reversine was purchased from Cayman Chemical Company and was kept as a 10 mM solution in DMSO.
7
Cell Lysis and Protein Extraction
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M2I-1 (ChemBridge Corporation), CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich, C3228), Protease inhibitor cocktail (Sigma-Aldrich, p8340), Reversine (C656820-32-5), MG132 (Cayman Chemical, 133407-82-6).
8
Cell Culture Conditions and Drug Treatments
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Cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Cells were grown at 37°C with 5% CO2 in a humidified environment. For experiments involving drug treatments, controls represent cells treated with vehicle alone. Reversine was obtained from Cayman Chemical, and Spautin-1 and BafA1 were purchased from Sigma-Aldrich.
9
Synchronization and Microtubule Disruption Assay
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293FT, JW36 HeLa, YFP-H2B HeLa, U2OS, and 6xHis-SUMO2 (a gift from Mary Dasso, National Institutes of Health, Bethesda, MD) cells were maintained at 37°C in a 5% CO2 atmosphere. Cells were grown in DMEM medium (Gibco) containing 10% fetal bovine serum (Atlanta Biologicals). Cells were not tested for mycoplasma contamination or authenticated otherwise. Thymidine (Sigma-Aldrich, St. Louis, MO.) was dissolved in DMSO and used at a final concentration of 2 mM; nocodazole (Calbiochem, Burlington, MA) was dissolved in DMSO and used at a final concentration of 100 ng/mL or 3.3 μM as indicated. To synchronize cells into S phase, cells were cultured in the presence of 2 mM Thymidine for 19 hr, released in drug-free media for 6 hr, and cultured in 2 mM Thymidine for 19 hr. For mitotic timecourse analysis, cells were released from the double Thymidine block and harvested in 2x SDS-sample buffer at indicated timepoints.
To weaken SAC signaling, 50 nM of the Mps1 kinase inhibitor, Reversine (Cayman Chemical, Ann Arbor, MI), was used immediately before timelapse acquisition.
To weaken SAC signaling, 50 nM of the Mps1 kinase inhibitor, Reversine (Cayman Chemical, Ann Arbor, MI), was used immediately before timelapse acquisition.
10
Pharmacological Inhibitors in Cell Research
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