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6 protocols using azd8055

1

Signaling Pathway Inhibitor Protocol

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The following antibodies and reagents were used in the present study: anti-AKT (#9272, Cell Signaling), anti-phospho-AKT (Ser473) (#4058, Cell Signaling), phospho-PDK1 (Ser241) (C49H2) (#3438, Cell Signaling), phospho-mTOR (Ser2448) (#5536, Cell Signaling), phospho-p70 S6Kinase (Thr389) (#9205, Cell Signaling), anti-GSK-3β (27C10) (#9315, Cell Signaling), p-Ser9-GSK-3-beta (#9322, Cell Signaling), anti-N-MYC(C-19) (SC-791, Santa Cruz Biotechnology), and anti- c-Myc (9E10) (sc-40, Santa Cruz Biotechnology).
MK-2206 (AKT inhibitor, A10003) was purchased from Selleckchem. GSK2334470 (PDK1 inhibitor, No. 4143) was purchased from Tocris Bioscience. AZD8055 (mTOR inhibitor, 1009298-09-2) was purchased from LC Laboratories. U0126 (MEK inhibitor, 70970-5) was purchased from Cayman Chemical.
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2

Pharmacological Inhibitors in Cancer Research

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Dasatinib, GDC-0941, AZD8055, rapamycin and MLN0128 were obtained from LC Laboratories (Woburn, MA); AKT inhibitor VIII from Chemdea (Ridgewood, NJ). InSolution Q-VD-OPh was from EMD Millipore (Billerica, MA); vincristine, doxycycline, etoposide, daunorubicin, MTX, 6-MP, dexamethasone and araC are from Sigma-Aldrich (St. Louis, MO). Palbociclib was purchased from Selleck Chemicals (Houston, TX).
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3

Small Molecule Inhibitor Preparation

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LY294002, Wortmannin (WM), AZD8055 were from LC Laboratories (Woburn, MA), Dexamethasone (Dex) and other chemicals were from Sigma-Aldrich (St. Louis, MO).
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4

Pharmacological Evaluation of Diverse Compounds

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Erlotinib, crizotinib, afatinib, vandetanib and AZD8055 were obtained from LC laboratories (Woburn, MA, USA), and BIBX 1382 was obtained from TOCRIS Bioscience (Bristol, UK). All other compounds were kindly provided by NCGC. Compounds used in this study are detailed in Table 1. Toxicity studies were performed to determine the maximal tolerated dose for in vivo studies. For in vitro studies, all compounds were dissolved in dimethyl sulfoxide (DMSO, MilliporeSigma, Burlington, MA, USA), and DMSO was used as a vehicle control. For in vivo studies, BIBX 1382, vandetanib, idarubicin, SAHA, staurosporine, and 17-AAG were dissolved in 10% DMSO, whereas crizotinib, imatinib, Erlotinib, afatinib, and AZD8055 were dissolved in 1X PBS. Either DMSO or PBS (Thermo Fisher Scientific, Waltham, MA, USA) was used as a vehicle control.
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5

Brassinosteroid Signaling Pathway Assay

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Sterilized Arabidopsis seeds were germinated and grown on solid ½ MS medium with DMSO, BL (Wako, 635–00811) or AZD8055 (LC Laboratories, A-2345) at the indicated concentrations in the light for 7 days. For BRZ response assays, sterilized seeds were plated on solid ½ MS medium with DMSO or the indicated concentrations of BRZ (kindly provided by Dr. Tadao Asami) [61 (link)]. Plates were exposed to light for 6–8 h and then kept in darkness for 7 days [8 (link)]. Plates were imaged using the Epson Perfection V600 Flatbed Photo scanner and hypocotyl lengths were measured using ImageJ [84 (link)].
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6

Autophagy Induction and Carbon Starvation Assay

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Seeds were surface-sterilized with 50%(v/v) bleach and then washed using sterile water. The seeds were germinated on 1× Murashige & Skoog (MS) solid medium [1× MS salt including vitamins (Duchefa, Haarlem, the Netherlands) with 1% (w/v) sucrose, 0.25% (w/v) phytagel (Sigma, St Louis, MO, USA)] or liquid medium with gentle shaking (100 rpm). Plants were incubated under long-day conditions (16 h light and 8 h dark) at 21-23°C.
To induce autophagy, the seedlings incubated in the liquid medium for 7 d were transferred to nitrogen-deficient medium (1 × MS micronutrient solution, 3 mM CaCl2, 1.5 mM MgSO4, 1.25 mM KH2PO4, 5 mM KCl and 1% sucrose, pH 5.7) and further incubated for indicated duration. For drug treatment, seedlings were treated with 0.5 µM AZD 8055 (LC Laboratories), 30 µM wortmannin (Sigma), and 0.5 µM Concanamycin A (Cayman).
Phenotypic analysis of the fixed carbon starvation response was performed as described (Chung et al., 2010 (link)). Briefly, after incubating seedlings on 1× MS-suc solid medium (1× MS salt including vitamins, 29 mM mannitol, and 0.25% phytagel) for 14 d, the seedlings were then deprived of light for 10 d. Seedlings resuming growth were counted after 18 d of recovery.
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