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134 protocols using selumetinib

1

Fibroblast-Melanoma Crosstalk Modulation

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Fully confluent fibroblasts plated in T162 flasks were treated overnight with either fresh media or fresh media supplemented with 100 ng/ml human recombinant IL-1β (PeproTech). The next morning, the cells were incubated in fresh media for 5 h, which was subsequently added to melanoma cells plated in either 6- or 12-well plates for 48 h with various inhibitors. The reagents used for these experiments were 1% DMSO (Sigma-Aldrich), 1 µM PLX4032 (Selleck Chemicals), 1 µM RAF265 (Selleck Chemicals), or 0.5 µM both PLX4032 and selumetinib (Selleck Chemicals). When the duotherapy treatment (PLX4032 and selumetinib) was also used in combination with either MK-2206, SB 225002, Bay 11-7082, or obatoclax, the concentration of each drug used was: 1 µM MK-2206 (Selleck Chemicals), 0.5 µM SB 225002 (Alfa Aesar), 0.2 µM Bay 11-7082 (Sigma-Aldrich), and 0.2 µM obatoclax (Selleck Chemicals). These concentrations were also used when these inhibitors were used as single agents. For each drug treatment, melanoma cells were cultured in nonconditioned media, conditioned media taken from unstimulated fibroblasts, or conditioned media taken from fibroblasts previously stimulated with IL-1β. Then, cell survival was assayed by crystal violet staining (outlined in the next section).
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2

Investigating Stress Response Signaling Pathways

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The following inhibitors were purchased from Selleck Chemicals, catalogue numbers as follows: GSK2656157 (hearefter referred to as PERKi), S7033; pictilisib (GDC-0941), S1065; AZD8055, S1555; apitolisib (GDC-0980), S2696; selumetinib (AZD6244), S1008; GDC-0879, S1104; RO5126766, S7170; SCH772984, S7101. AKT inhibitor VIII (AKTi VIII) was purchased from Merck Millipore, catalogue number 124018. Concentrations were selected from a dilution series performed to ensure activity. Concentrations used for all experiments are shown in Table 1. Cells were treated with inhibitor 1 h prior to induction of ER-stress.

Inhibitors, major targets and concentrations used in this study.

Table 1
InhibitorMajor target(s)ConcentrationReference
GSK2656157PERK50 and 500 nM[42 (link)]
pictilisib (GDC-0941)PI3K500 nM[43 (link)]
AKT inhibitor VIIIAKT PH domain5 μM[44 (link)]
AZD8055MTOR500 nM[45 (link)]
apitolisib (GDC-0980)PI3K and MTOR500 nM[46 (link)]
selumetinib (AZD6244)MEK150 nM[47 (link)]
GDC-0879BRAF/CRAF50 nM[48 (link)]
RO5126766BRAF/CRAF/MEK50 nM[49 (link)]
SCH772984ERK1/250 nM[50 (link)]
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3

Isolation and Culture of Mouse Cortical Neurons

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Mouse cortical neurons were prepared from E16.5 FVB/N mice and plated in poly-D-lysine coated 12-well plates (5×105 per well) in Neurobasal medium supplemented with GlutaMAX (Invitrogen), B-27, antibiotics (Penicillin/Streptomycin). Half of the media was changed with fresh media every 3–4 days. For drug treatment, DMSO (Sigma), pimasertib (2 μM, Selleckchem), or selumetinib (0.4 μM, Selleckchem) was applied to the media starting at days in vitro (DIV) 1. For shRNA virus infection experiments, neurons were infected with lentiviruses at DIV 1 with the multiplicity of infection at 10. Neurons were harvested at DIV 15–18 and lysed with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with phosphatase and protease inhibitors (GenDEPOT). Lysates were cleared by centrifugation (20 min, 15,000 rpm, 4°C) followed by protein quantification via BCA assay (Pierce). LDS Sample buffer (Invitrogen) with a reducing agent was added to each lysate followed by a 10 min incubation at 95°C. Samples were spun down and electrophoresed on a 4–12% Bis-Tris gel, transferred to a nitrocellulose membrane and blocked for one hour with 5% non-fat milk prior to primary antibody incubation. Unless otherwise indicated, the samples used for western blot were from whole lysates.
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4

Preparation of Inhibitor Stock Solutions

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Vemurafenib (ChemieTek) was dissolved in dimethyl sulphoxide (DMSO; Sigma-Aldrich) and stored at –20° C at stocks of 100 mM. Leflunomide (Sigma-Aldrich) was dissolved in DMSO and stored at 4° C at stocks of 10 mM. AZD6244 (selumetinib; SelleckChem) was dissolved in DMSO and stored at –20° C at stocks of 2 mM. When aliquots of the stock were in use they were stored at 4° C for no longer than two weeks.
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5

MEKi Culturing of Naive hESCs

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The following MEKi were used to culture naïve hESCs at the concentrations indicated in the manuscript: PD0325901 (Axon Medchem), Selumetinib (Selleck Chemicals), Binimetinib (Selleck Chemicals), Trametinib (Selleck Chemicals), Pimasertib (Selleck Chemicals), Refametinib (Selleck Chemicals), TAK-733 (Selleck Chemicals), RO5126766 (Selleck Chemicals), Cobimetinib (Selleck Chemicals), AZD8330 (Selleck Chemicals).
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6

Chemical Procurement for Research

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All non-drug chemicals were purchased from Sigma Aldrich (St. Louis, MO). Solvents were purchased from Acros organics (Morris Plains, NJ). All drugs were purchased from LC-Laboratories (Woburn, MA), except glibenclamide, glimepiride, and cyclosporine which were purchased from Sigma Aldrich, TAK632 from AdooQ Bioscience (Irvine, CA), and talazoparib, fulvestrant, venetoclax, selumetinib, and taselisib from Selleckchem (Houston, TX).
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7

Inhibition of Receptor Tyrosine Kinases

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Trametinib (GSK1120212), selumetinib (AZD6244), lapatinib, crizotinib and MK2206 were purchased from Selleck Chemicals (Houston, TX). Recombinant human NRG1, PDGF-BB, and IGF-1 were purchased from Cell Signaling Technology (Beverly, MA); recombinant human EGF was purchased from Lonza Walkersville Inc141 (Walkersville, MD); recombinant human HGF was provided by Michael P. Lisanti (University of Manchester, UK). Humanized ERBB3 monoclonal antibody U3-1287 was provided by U3 Pharma (Martinsried, Germany).
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8

Selumetinib Efficacy in KRAS-Driven Tumors

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We injected 106 NIH3T3 cells expressing KRASG12C or KRASG12D into 6-week-old female SHO mice (Crl:SHO-PrkdcscidHrhr; Charles River Laboratories). Treatments were started when tumor volume reached approximately 250 mm3. Selumetinib (AZD6244; ARRY-142886) was purchased from Selleckchem (S1008) and dissolved in 0.5% methylcellulose with 0.4% polysorbate-80. Tumor-bearing mice were administered 25 mg/kg Selumetinib by oral gavage twice daily, and tumor volume was measured and calculated as length × width × width/2.
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9

Establishment and Characterization of Entrectinib-Resistant Cell Line

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HCC78 cell was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Cell line identity was authenticated by short-tandem-repeat analysis. Entrectinib-resistant HCC78 (HCC78ER) cells were newly established in our laboratory through the exposure of HCC78 cells to gradually increasing concentrations of Entrectinib (starting at 100 nM and ending with 5 μM) over 6 months. The established cells maintained resistance to Entrectinib even after the withdrawal of Entrectinib from the culture medium.
Entrectinib was provided by Ignyta, Inc./F.Hoffmann-La Roche Ltd. Crizotinib, ceritinib, lorlatinib and selumetinib were purchased from Selleckchem. All drugs were dissolved at a 10 mM concentration in dimethyl sulfoxide (DMSO) and stored in small aliquots at −20 °C until further use. Antibodies specific for p-ROS1 (Tyr1068), ROS1, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-STAT3 (Tyr705), STAT3, p-p53 (Ser15), p53, p-H2AX (Ser139), H2AX and PARP were obtained from Cell Signaling Technologies. Anti-FGF3 and β-actin antibodies were obtained from Santa Cruz Biotechnology.
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10

Selumetinib Inhibits Tumor Growth in LLC Mouse Model

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All experimental animal protocols were approved by and used in compliance with the Indiana University School of Medicine Institutional Animal Care and Use Committee. Eight-week old male C57BL/6J mice were obtained from The Jackson Laboratory. All mice were maintained on a regular light-dark cycle and allowed free access to food and water throughout the duration of the experiment. Mice were grouped as follows: Control + vehicle (n = 6), LLC + vehicle (n = 8), and LLC + Selumetinib (n = 8). Tumor bearing mice were subcutaneously injected with 106 LLC cells in the intrascapular region on day 0, with treatments beginning 24 h later. Selumetinib (Selleckchem) in vehicle (0.5% methylcellulose/0.2% Tween 80) or vehicle alone was administered twice daily at 25 mg/kg by gavage (Shannon et al., 2009 (link); Troiani et al., 2012 (link); Huang et al., 2013 (link)). Body weights of the mice were recorded daily. Mice were euthanized under general anesthesia on day 17 when some mice reached the criteria for a humane endpoint. Muscles, tumors and organs were dissected, weighed, snap frozen in liquid nitrogen, and stored at −80°C. Tissue weights are expressed as a percentage of initial body weight to normalize for small differences in starting size.
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