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.
Az3146
Manufactured by Bio-Techne
Sourced in United Kingdom
AZ3146 is a small molecule inhibitor designed for research purposes. It targets the Aurora kinase family, which play a role in cell division and proliferation. The product is intended for laboratory use only and should not be used for diagnostic or therapeutic applications.
Automatically generated - may contain errors
Lab products found in correlation
Nocodazole, by Merck Group (6 mentions)
Mg132, by Merck Group (4 mentions)
Reversine, by Cayman Chemical (2 mentions)
Gsk923295, by Bio-Techne (2 mentions)
Thymidine, by Merck Group (2 mentions)
Reversine, by Merck Group (2 mentions)
Paclitaxel, by Merck Group (2 mentions)
Okadaic acid, by Santa Cruz Biotechnology (2 mentions)
Icrf 193, by Santa Cruz Biotechnology (2 mentions)
S trityl l cysteine, by Merck Group (2 mentions)
On targetplus sirna, by Horizon Discovery (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Promega (2 mentions)
Bi2536, by Thermo Fisher Scientific (2 mentions)
Mg132, by Selleck Chemicals (2 mentions)
Nocodazole, by Cayman Chemical (1 mentions)
Mg132, by Bio-Techne (1 mentions)
D8418, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Bafa1, by Merck Group (1 mentions)
15 protocols using az3146
1
Cell Cycle Arrest and Silencing Assays
2
Mitotic Arrest Protocols and Inhibitor Treatments
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HeLa cells were arrested in mitosis using either 100 ng/ml nocodazole (487928; Merck) or 10 µM STLC (164739-5G; Merck) for 2.5 h (immunofluorescence) or 18 h (immunoblotting).
For biochemical analysis, mitotic cells were harvested by shake-off, washed twice in PBS and once in Opti-MEM, both of which had been pre-equilibrated to 37°C, 5% CO2. Cells were counted and resuspended in pre-equilibrated Opti-MEM to give 7.5 × 106 cells/ml. Subsequently, cells were either treated with 0.5 µM Aurora A inhibitor MLN8237 (A4110-APE-10 mM; ApexBio), 10 µM Aurora B inhibitor ZM 447439 (A4113-APE-10 mM; ApexBio), 10 µM Aurora B inhibitor AZD1152 (A4112-APE-10 mM; ApexBio), 25–100 nM PP1/PP2A inhibitor calyculin A (1,336/100 U; TOCRIS), and 40 µM proteasome inhibitor MG-132 (474790-5MG; Merck) along with 1–10 µM MPS1 inhibitor (AZ3146; TOCRIS) or DMSO (D8418; Merck) for up to 25 min.
For immunofluorescence microscopy, nocodazole- or STLC-arrested cells were treated with 20 µM MG132 and the inhibitors described above for 30 min prior to fixation. For the CENP-E inhibitor experiment, asynchronous cultures were treated with MG132 for 3 h, or 300 nM CENP-E inhibitor (GSK923295; TOCRIS) for 3 h and MG-132 with or without Aurora A inhibitor for 30 min before fixation.
For biochemical analysis, mitotic cells were harvested by shake-off, washed twice in PBS and once in Opti-MEM, both of which had been pre-equilibrated to 37°C, 5% CO2. Cells were counted and resuspended in pre-equilibrated Opti-MEM to give 7.5 × 106 cells/ml. Subsequently, cells were either treated with 0.5 µM Aurora A inhibitor MLN8237 (A4110-APE-10 mM; ApexBio), 10 µM Aurora B inhibitor ZM 447439 (A4113-APE-10 mM; ApexBio), 10 µM Aurora B inhibitor AZD1152 (A4112-APE-10 mM; ApexBio), 25–100 nM PP1/PP2A inhibitor calyculin A (1,336/100 U; TOCRIS), and 40 µM proteasome inhibitor MG-132 (474790-5MG; Merck) along with 1–10 µM MPS1 inhibitor (AZ3146; TOCRIS) or DMSO (D8418; Merck) for up to 25 min.
For immunofluorescence microscopy, nocodazole- or STLC-arrested cells were treated with 20 µM MG132 and the inhibitors described above for 30 min prior to fixation. For the CENP-E inhibitor experiment, asynchronous cultures were treated with MG132 for 3 h, or 300 nM CENP-E inhibitor (GSK923295; TOCRIS) for 3 h and MG-132 with or without Aurora A inhibitor for 30 min before fixation.
3
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.
4
Molecular Toolkit for Cell Biology Research
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MG132, paclitaxel, nocodazole, and reversine were purchased from Sigma-Aldrich (St. Louis, MO) and stocked as a 10 mM solution in dimethyl sulfoxide (DMSO). AZ 3146 was obtained from Tocris (Bristol, United Kingdom) and stocked as a 10 mM solution in DMSO. The pan-caspase inhibitor z-VAD-fmk was obtained from Bachem Bioscience (Bubendorf, Switzerland) and stocked as a 50 mM solution in N,N-dimethylformamide (DMF). The appropriate amount of DMSO and/or DMF was always employed for negative control conditions.
5
Cell Cycle Regulation Assay Protocol
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Where indicated, cells were incubated in 1 μM ICRF193 (Santa Cruz), 1 μM Okadaic acid (Santa Cruz) or 2 μM AZ3146 (Tocris) for 5 min or 10 μM S-trityl-L-cysteine (Sigma) for 20 minutes (Figure 2E, F ) or 2h (Figure S3C ) before imaging. For RNAi, cells were transfected with 50 nM ON-TARGET plus siRNAs (Dharmacon) targeting RAD21 (AUACCUUCUUGCAGACUGUUU), KIF18A (GCCAAUUCUUCGUAGUUUU), Ska1 (pool targeting: GGACUUACUCGUUAUGUUA, UCAAUGGUGUUCCUUCGUA, UAUAGUGGAAGCUGACAUA and CCGCUUAACCUAUAAUCAA), or a nontargeting control using Lipofectamine RNAi MAX (Invitrogen) following instructions of the manufacturer. Plasmids containing wild type cyclin B1-mCherry or the non-degradable mutant (R42A and L45A) (Gavet and Pines, 2010 (link); Vazquez-Novelle et al., 2014 (link)) were transfected into HeLa cells using Fugene HD (Promega) according to manufacturer’s instructions 24 hours prior to imaging.
6
Assessing Small Molecule Inhibitors
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All chemicals were resuspended in DMSO. NSC697923 (Sigma-Aldrich) was used at 20 µM. Nocodazole (Sigma-Aldrich) was used at 50 nM. MG132 (EMD Biosciences) was used at 10 µM. CC0651 (Thermo Fisher Scientific) was used at 50 µM. Taxol (Life Technologies) was used at 1 µM. PYR-41 (Santa Cruz Biotechnology) was used at 20 µM. TAK-243 (formerly MLN7243) was a gift from Hidde Ploegh (Whitehead Institute) and used at 25 µM. PR-619 (Sigma-Aldrich) was used at 100 µM. ZM447439 (R&D Systems) was used at 2 µM. VX680 (LC Laboratories) was used at 2.5 µM. Flavopiridol (Santa Cruz Biotechnology) was used at 5 µM. AZ3146 (Tocris) was used at 2 µM. BI2536 (Thermo Fisher Scientific) was used at 10 µM. Okadaic acid (VWR) was used at 1 µM. To dose cells, drugs were diluted in fresh media and then added to the cells. For immunofluorescence, cells were dosed for 15 min before fixation.
7
High-Throughput Screening of Mitotic Regulators
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The drugs used for screening in this study are listed in Supplementary Table 1 .
Reversine, SP600125, ZM 447439, BI 2536, STLC, Dimethylenastron, SB203508, RO 3306, Cdk1 Inhibitor III, Roscovitine, NSC 95397, IPA3, Y-27632, ITX-3, Nocodazole, Paclitaxel, Blebbistatin, Cytochalasin B, MG 132 and Velcade were purchased from Sigma–Aldrich (St. Louis, MO, USA). AZ 3146 and Mps-BAY2a were obtained from Tocris (Bristol, United Kingdom). MLN8237 (Alisertib), AZD1152-HQPA (Baraserib) and SB743921 were purchased from Selleckchem. GSK923295 is from MedChem express.
Monoclonal antibodies against α-tubulin, CDK1, AURKA, PLK1, KIF11 (Sigma-Aldrich); CCNB1, CCNE1, TP53, CDKN1A, JNK1, JNK2 (Cell signaling); EB1, COFILIN (Santa Cruz), TTK, BUBR1 (Abcam). Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [69 (link)]; Kif23 [70 (link)] were used.
Reversine, SP600125, ZM 447439, BI 2536, STLC, Dimethylenastron, SB203508, RO 3306, Cdk1 Inhibitor III, Roscovitine, NSC 95397, IPA3, Y-27632, ITX-3, Nocodazole, Paclitaxel, Blebbistatin, Cytochalasin B, MG 132 and Velcade were purchased from Sigma–Aldrich (St. Louis, MO, USA). AZ 3146 and Mps-BAY2a were obtained from Tocris (Bristol, United Kingdom). MLN8237 (Alisertib), AZD1152-HQPA (Baraserib) and SB743921 were purchased from Selleckchem. GSK923295 is from MedChem express.
Monoclonal antibodies against α-tubulin, CDK1, AURKA, PLK1, KIF11 (Sigma-Aldrich); CCNB1, CCNE1, TP53, CDKN1A, JNK1, JNK2 (Cell signaling); EB1, COFILIN (Santa Cruz), TTK, BUBR1 (Abcam). Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [69 (link)]; Kif23 [70 (link)] were used.
8
Cell Culture and Small Molecule Inhibitors
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DLD-1, HeLa, RKO and HCT-116 cell lines were cultured in DMEM plus 10% fetal calf serum (LifeTechnologies), 2 mM glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (Lonza) at 37°C in a humidified 5% CO2 atmosphere. DLD-1 Histone-H2B-mCherry were as described previously [62 (link)]. Small molecule inhibitors dissolved in DMSO were as follows: GSK923295, Cenp-E inhibitor (in house); AZ3146, Mps1 inhibitor (Tocris); Monastrol, Eg5 inhibitor (Sigma).
9
Concentration-Dependent SAC Inhibitor Assay
10
Cell Cycle Regulation Assay Protocol
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