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9 protocols using GDC-0941

The culture medium was Stem Span SFEM medium (Stem Cell Technologies, Vancouver, BC, Canada). NSC95397 and BN82002 (Calbiochem; Merck Millipore, Darmstadt, Germany), as well as the trial compounds ALX1, ALX2, ALX3, and ALX4, were examined at a final concentration of 10 μM. This concentration has been used in previous studies for NSC95397 and BN82002 [38 (link),58 (link)]. For the trial compounds, we additionally performed dose-response examinations (100, 50, 25, 10, 5, 2.5, and 1 μM) of their effects on cytokine-dependent AML cell proliferation (four patients examined together with the AML cell lines KG-1a, CTV-1, EOL, and HL60), and at 10 μM, the antiproliferative effects of these drugs (as well as NSC95397 and BN82002) had reached a plateau for those patient cells/AML cell lines that were susceptible to CDC25 inhibition. This concentration was therefore used in our experiments to detect differences among cells/patients in the susceptibility to growth inhibition induced by CDC25 inhibition. GDC0941 (Axon Medchem, Groningen, The Netherlands) and rapamycin (Sigma Aldrich, St. Louis, MO, USA) were used at a final concentration of 0.1 μM; this concentration can also be used to detect differences among patients [40 (link)].
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In order to check whether the regulation of DEK is cell cycle dependent, UACC-62 cells were treated with BRAF inhibitor vemurafenib (10 μM, Selleckchem, S1267), MEK inhibitor U0126 (5 μM, Calbiochem, 662005), PI3K inhibitor GDC0941 (Axon Medchem, 1377) or DMSO as a solvent control (1:1000, Merck, 103562) for 8 h or 24 h.
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LY294002 was from Sigma-Aldrich (St. Louis, MO, USA), GDC-0941 and CT98014 were from Axon Medchem (Groningen, Netherlands), Annexin-V-FITC was generated in our lab. Annexin-V-APC (RUO) was from Becton Dickinson (Franklin Lakes, NJ, USA). 4-OHT was from Sigma-Aldrich (St. Louis, MO, USA).
IL-2 and IL-3 were from Peprotech (Rocky Hill, NJ, USA).
The following antibodies were used for western blotting: Puma (#3043) was from Prosci (Poway, CA, USA), human Puma (#12450), Foxo3a (#2497) were from Cell Signaling Technologies (Danvers, MA, USA). GSK3a/b (sc-56913), 14-3-3 (sc-1657) and NFATc1 (sc-7294) were from Santa Cruz (Dallas, TX, USA). Tubulin (MCA77G) was from Bio-Rad (Hercules, CA, USA). MCL1 (600-401-394S) was from Rockland (Limerick, PA, USA).
FLAG-M2 agarose affinity beads were from Sigma-Aldrich (St. Louis, MO, USA).
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The class I PI3K inhibitor, GDC-0941 (Axon Medchem), was used to treat bladder cancer cell lines and TERT-NHUC. The dose range chosen was based on previous studies that report IC50 values of 0.28–0.95 μM for cell viability of solid tumor cell lines, as well as pharmacokinetic data available from phase I clinical studies that report a maximum of 2 μM GDC-0941 plasma concentration in patients [30 (link), 41 (link)]. Cell viability was assessed by CellTiter-Blue® (Promega) analysis of bladder cancer cell lines and TERT-NHUC subjected to GDC-concentrations from 0 to 2 μM. Cell viability was assessed by CellTiter-Blue® (Promega) analysis. 1000–4000 cells per well (number of cells determined to ensure that confluence was not achieved in untreated controls during the experiment) were plated in 96-well plates in five replicate wells and allowed to attach for 24 h before addition of 0–2 μM GDC-0941 in 0.1 % DMSO. After 72 h, 20 μl of CellTiter-Blue solution was added to the medium for 2 h and fluorescence read at 550 nm. Medium alone was used as a blank. Prism software (GraphPad Software, La Jolla, CA, USE) was used to calculate IC50 values.
Cell cycle and apoptosis analysis of cells cultured with 1 μM GDC-0941 or DMSO only for 48 h was evaluated by flow cytometry as described [40 (link)]. All assays were done in triplicate and repeated at least three times.
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The MEK inhibitor PD184352 (in short PD) and GDC-0941 were purchased from Axon Medchem and Selleckchem, respectively. The PI3K inhibitor LY294002, the EGFR inhibitor AG1478 and cisplatin were obtained from Calbiochem. ON-TARGET plus SMART pool siRNA for BBC3 (PUMA), SPRED1, SPRED2 and non-targeting siRNA were from Thermo Scientific. For transfection of siRNA, X-tremeGENE siRNA Transfection Reagent (Roche) was used according to the manufacturer's instructions.
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Reagents were from Sigma (St. Louis, MO) unless noted. Iodoacetate, lactate, pyruvate, and cycloheximide stocks were prepared in water. Rotenone, oligomycin A, and AICAR were dissolved in DMSO. BEZ235 (Axon Medchem, Reston, VA), Torin-1 (Tocris Bioscience, Bristol, UK), Torin-2 (Selleck, Houston, TX), BKM120 (Axon Medchem), GDC0941 (Axon Medchem), Gefitinib (Axon Medchem), MK2206 (Selleck), PD 0325901 (Sigma and Selleck), and Rad001 (SU2C PI3K Dream Team Mouse Pharmacy, which obtains compounds from Shanghai Haoyuan Chemexpress; [Elkabets et al., 2013 (link)]) were dissolved in DMSO. For GF titration, EGF (Peprotech, Rocky Hill, NJ) and insulin (Sigma) were diluted in PBS and added at indicated concentrations.
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Protein G–Sepharose was from GE Healthcare. [γ-32P]ATP was from PerkinElmer. Agarose-conjugated anti-FLAG M2 antibody, Triton X-100, EDTA, EGTA, sodium orthovanadate, sodium glycerophosphate, sodium fluoride, sodium pyrophosphate, 2-mercaptoethanol, sucrose, benzamidine, Tween 20, Tris/HCl, sodium chloride, magnesium acetate and doxycyclin were from Sigma. PMSF was from Melford. Tissue culture reagents, Novex 4–12% Bis-Tris gels and NuPAGE LDS sample buffer was from Invitrogen. Ampicillin was from Merck. P81 phosphocellulose paper was from Whatman. Methanol and chloroform were from VWR Chemicals. Inhibitors GDC-0941 (Axon Medchem), GSK2334470 (Tocris), AZD8055 (Selleck) and BKM120 (Chemie Tek) were purchased from the indicated suppliers. VPS34-IN1 (1-[{2-[(2-chloropyridin-4yl)amino]-4′-(cyclopropylmethyl)-[4,5′-bipyrimidin]-2′-yl}amino]-2-methyl-propan-2-ol) was synthesized as described in patent WO 2012085815 A1 [Cornella Taracido, I., Harrington, E.M., Honda, A. and Keaney, E. (2012) Preparation of bipyrimidinamine derivatives for use as Vps34 inhibitors; method for synthesis of this compound is described on page 73, Table 4, example 16a.VPS34-IN1 has a CAS registry number 1383716-33-3].
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The following inhibitors were obtained from Selleck Chem (Munich, Germany, unless otherwise indicated): JAK inh 1 (Merck Millipore, Billerica, MA, USA; #420099), ruxolitinib (#S1378), pimozide (Sigma-Aldrich, Zwijndrecht, The Netherlands; #P1793), Ly294002 (Cell Signaling Technology, Leiden, The Netherlands; #9901), MK-2206 (#S1078), rapamycin (#S1039), CI-1040 (Axon Medchem, Groningen, The Netherlands; #1368), AZD6244 (#S1008) and GDC-0941 (#S1065).
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Cells were plated in 96-well plates at a density of 5,000 cells/well for Tera, TeraCP, and 833KE, and 7,000 cells/well for Scha and NCCIT. At 2 to 3 hours after plating cells, one of the following agents was added, cisplatin (Accord Healthcare), Everolimus (Santa Cruz Biotechnology), GDC-0941, MK-2206, AZD8055, MLN0128 (also known as TAK-228; all from Axon Medchem), and dasatininb (Selleckchem). After a 96-hour incubation, 3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium (MTT; Sigma) was added at a concentration of 5 mg/mL for 4 hours. Medium was removed and the formazan crystals were dissolved in DMSO (Sigma). Absorbance was measured at 520 nm using an iMARK microplate absorbance reader (Bio-Rad). Relative survival was determined as the decrease in signal compared with untreated cells.
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