HeLa, DLD-1, BT-549, HCC1937, KB2P1.21 and KB2P1.21R1 tumour cell lines were plated in 96-wells plates. BT-549 cells were pre-treated with 1 μg ml−1 doxycycline for 48 h. HeLa were plated at 2,000 cells per well, DLD-1 cells at 5,000 cells per well, BT-549 and HCC1937 cells at 1,000 cells per well, and KB2P1.21 and KB2P1.21R1 were plated at 1,200 cells per well. Cells were allowed to attach for 3 or 24 h and were treated with indicated concentrations of olaparib, MK-1775, or MK4827 (all from Axon Medchem, Groningen, the Netherlands) for 3 or 4 days. Methyl-thiazol tetrazolium (MTT) was added to cells at a concentration of 5 mg ml−1 for 4 h, after which culture medium was removed and formazan crystals were dissolved in DMSO. Absorbance values were determined using a Bio‐Rad benchmark III Biorad microtiter spectrophotometer at a wavelength of 520 nm. Proliferation was determined as the relative decrease in signal compared to DMSO-treated cells. Unless mentioned otherwise, statistical significance was tested using Student’s t-test.
Mk 1775
Manufactured by Axon Medchem
Sourced in Netherlands
MK-1775 is a laboratory equipment product. It is a small molecule that inhibits the activity of the Wee1 kinase, which plays a role in cell cycle regulation. The core function of MK-1775 is to serve as a research tool for studying Wee1 kinase and its role in cellular processes. No further details are provided to avoid extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark 3 biorad microtiter spectrophotometer, by Bio-Rad (1 mentions)
Olaparib, by Axon Medchem (1 mentions)
Compusyn software 1, by ComboSyn (1 mentions)
Gsk 923295, by Merck Group (1 mentions)
Vinblastine, by Bio-Techne (1 mentions)
Bi2536, by Axon Medchem (1 mentions)
Hydroxyurea, by Merck Group (1 mentions)
Ro 3306, by Bio-Techne (1 mentions)
β actin a5441, by Merck Group (1 mentions)
Paclitaxel, by Chemietek (1 mentions)
Pf 00477736, by Pfizer (1 mentions)
Mts assay, by Promega (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Olaparib, by LC Laboratories (1 mentions)
Infinite m200, by Tecan (1 mentions)
Ku55933, by Axon Medchem (1 mentions)
Phospho cdk1 tyr15, by Cell Signaling Technology (1 mentions)
Wee1 d10d2, by Cell Signaling Technology (1 mentions)
Vinculin hvin 1, by Merck Group (1 mentions)
Mg132, by Santa Cruz Biotechnology (1 mentions)
7 protocols using mk 1775
1
Cytotoxicity Assay of Tumor Cell Lines
2
Synergistic Effects of dl922-947 and AZD1775 on MM Cells
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MM cells were seeded in 96-well plates 24 h before treatment with dl922-947 and AZD1775 (MK1775, purchased from Axon Medchem), both alone and in combination at various concentrations in a constant ratio. Five days after treatment, cells were fixed with 50% (v/v) trichloroacetic acid and stained with 0.4% (w/v) SRB in 1% (v/v) acetic acid, following the manufacturer’s instructions. Synergism, additivity, or antagonism were determined through isobologram analysis using the CompuSyn software 1.0 (ComboSyn, Inc., Paramus, NJ, USA). Combination index (CI) values were also calculated by the CompuSyn software, which uses the Chou–Talalay method. CI < 1 indicates synergism, CI = 1 additivity, and CI > 1 antagonism. The r value represents the linear correlation coefficient of the median effect plot, which indicates the conformity of the data to the mass action law.
3
Inhibitor Sources for Cell Studies
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Inhibitors were purchased from the following distributors: AdooQ BioScience (Irvine, CA, USA; Aurora A inhibitor I, BI‐2536, epothilone A, MK‐1775 and VX‐680); Axon Medchem (Reston, VA, USA; SPL‐B); Focus Biomolecules (Plymouth Meeting, PA, USA; Latrunculin A); MedChemExpress (Princeton, NJ, USA; PF‐2771); Selleck Chemicals (Houston, TX, USA; GSK‐923295); Sigma‐Aldrich (Importazole, RO‐3306, and S‐trityl‐l ‐cysteine); Thermo Fisher Scientific (Waltham, MA, USA; Colcemid (KaryoMAX Colcemid)); Calbiochem (San Diego, California CA, USA; Etoposide); LKT Laboratories (St. Paul, MN, USA; Vinblastine); Tocris Bioscience, (Bristol, UK; Monastrol); Wako (Cytochalasin B, daunorubicin, doxorubicin, nocodazole and paclitaxel).
4
Evaluating WEE1 Inhibitor MK-1775 Effects
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The small molecule WEE1 inhibitor, MK-1775, was purchased from Axon Medchem (Groninberg, Netherlands) or synthesized by us. Dimethyl sulfoxide (DMSO) was used to reconstitute the drug and subsequent aliquots were stored in a desiccator at -20°C. An equivalent amount of DMSO for the highest concentration of drug was used for each experiment as a vehicle control.
5
Cell Cycle Regulation Assay Protocol
6
Cytotoxicity Assay for Drug Screening
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Cisplatin was purchased from Sigma Aldrich; paclitaxel from ChemieTek; olaparib from LC Laboratories; VE822, MK1775 and KU55933 from Axon Medchem; THZ1 and THZ1 hydrochloride were from Insight Biotechnology Limited; ET-743 from PharmaMar and PF-00477736 from Pfizer. For cytotoxicity experiments, cell lines were seeded in 96-well plates and were treated after 48 hours with different concentrations of the drugs. After 72 hours cell viability was examined with the MTS assay (Promega) and absorbance was acquired using a plate reader (Infinite M200, TECAN). Drug concentrations inhibiting growth in 50% of the cells (IC50s) were calculated for each cell line, with the interpolation method.
7
Antibody panel for cell analysis
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The following commercial antibodies were used at the indicated concentrations for Western blot (WB) and immunofluorescence (IF): a-tubulin (DM1A, Sigma-Aldrich; Cat#T6199, 1:1000 IF), Ki67 (Abcam; Cat#ab15580; 1:1000 IF), E-cadherin (DECMA-1, GeneTex; Cat#GTX11512, 1:1000 IF), aE-catenin (Enzo; Cat#ALX-804-101-C100, 1:500 IF), a-catenin (Sigma-Aldrich; Cat#C2081, 1:500 IF), Phospho-Histone H3 Ser10 (3H10, Millipore; Cat#05-806, 1:1000 IF), Wee1 (D10D2, Cell signaling; Cat#13084, 1:500 IF, 1:1000 WB), a-actinin (BM-75.2, Sigma-Aldrich; Cat#A5044, 1:500 IF), phospho-Cdk1 Tyr15 (Cell signaling; Cat#9111, 1:250 IF), Vinculin (hVIN-1, Sigma-Aldrich; Cat#V9131, 1:250 IF), Cdk1 (PSTAIR; Millipore; Cat#06-923; 1:500), Cdk1 (POH1, Cell signaling; Cat#9116, 1:250 IF), p130CAS (BD Biosciences; Cat#610271, 1:3000 WB), phospho-Histone H2A.X Ser139 (JBW301; Millipore; Cat#05-636, 1:1000 IF).
The following reagents were used at the indicated concentrations: MK-1775 (Axon Medchem; Cat#1494, 500 nM), Calyculin A (Enzo; Cat#BML-EI192-0100, 10 ng/mL), MG132 (Santa Cruz; Cat#sc-201270, 5mM), Thapsigargin (Sigma-Aldrich; Cat#T9033, 1 mM), Yoda (Tocris; Cat#5586, 10 mM).
The following reagents were used at the indicated concentrations: MK-1775 (Axon Medchem; Cat#1494, 500 nM), Calyculin A (Enzo; Cat#BML-EI192-0100, 10 ng/mL), MG132 (Santa Cruz; Cat#sc-201270, 5mM), Thapsigargin (Sigma-Aldrich; Cat#T9033, 1 mM), Yoda (Tocris; Cat#5586, 10 mM).
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