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16 protocols using vemurafenib

1

Generating Vemurafenib-Resistant BCPAP Cells

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In order to create vemurafenib-resistant BCPAP cells, cells were treated for 24 hours with increasing concentrations of vemurafenib (Chemietek, Indianapolis, IN) up to 16 μM, subcultured and allowed to grow to 70% confluence before the next treatment. Cells were treated with vemurafenib concentrations of 10 μM, 12 μM, 14 μM, and 16 μM over a period of 45 days. The resistant BCPAP cells were maintained in culture with intermittent treatments of 16 μM vemurafenib.
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2

Preparation of Vemurafenib and CCT196969

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CCT196969 and vemurafenib were purchased from ChemieTek (Indianapolis, IN, USA). Both drugs were dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich Inc.) at a stock concentration of 50 mM and stored as aliquots at -20°C until use.
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3

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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4

Melanoma Cell Line Characterization

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The following human melanoma cell lines were used and acquired at the American Tissue Type Collection: WM266.4 (BRAFV600D/RASWT), SKMEL28 (BRAFV600E/RASWT, STR profiled in house (LGC Standards, UK) on the 16th October 2015) and CHL-1 (BRAFWT/RASWT). D04 (BRAFWT/RASQ61L) cells were a kind gift from Dr. Amine Sadok and were tested by STR profiling on the 13th June 2014. Vemurafenib and 13C-glucose were purchased from Chemietek (Indianapolis, USA) and Sigma-Aldrich (Gillingham, UK), respectively.
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5

Cell Cycle and Colony Formation Assays

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Assays for colony formation and cell cycle analysis are described in Supplementary Methods (available online). Cells were treated with varying concentrations of vemurafenib for 72 hours or dabrafenib (Chemietek, Indianapolis, IN) for 48 hours or as indicated. Dimethyl sulfoxide was used as a vehicle.
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6

Synthesis and Use of Ubiquitin Probes

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EOAI3402143 (referred to as G9) was synthesized and provided by Evotec (UK), (Abingdon Oxfordshire, UK). Other reagents used in this study were obtained from the following sources: hemagglutinin-tagged ubiquitin vinyl methyl sulfone (HA-UbVS; Boston Biochem); Vemurafenib (PLX4032; Chemie Tek); PD0325901 (Cayman Chemical); MI-219 (A kind gift of Dr. Shaomeng Wang, University of Michigan). All reagents were made up and stored frozen as 10 mM stock solutions.
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7

Modulating Tumor Growth with Dietary Copper

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5 × 106 MEFs or 107 melanoma cells resuspended in phosphate buffer saline were injected subcutaneously into flanks of SCID/beige mice (Charles River Laboratory) as previously described42 (link). Drug treatments were as follows: vehicle [1% methylcellulose (Sigma), 1% dimethyl sulfoxide (DMSO, Sigma)], 2.0 mg tetrathiomolybdate (TTM, Sigma) in vehicle, or 20 mg/kg vemurafenib (Chemietek) in vehicle every other day (q.o.d) via oral gavage. Mice were fed a normal diet (PMI 5053 Picolab Mouse Diet 20, LabDiet) or where indicated, a Cu-deficient diet (CuD diet, TD.80388, Harlan Teklad). Mice fed a CuD diet mice were administered deionized H2O (diH2O) supplemented with 20 mg/L CuSO4, diH2O alone, or diH2O supplemented with 3 g/L trientine dihydrocholoride (Sigma). All studies were approved by the Duke University Institutional Animal Care and Use Committee. Statistical analysis of tumor volumes at end point was performed using a one-tailed, unpaired T-test, with a 95% confidence interval for two group datasets or one-way analysis of variance (ANOVA) with a 95%, 99%, or 99.9% confidence interval and Tukey’s multiple comparison post test for ≥ 3 datasets in Prism 5 (GraphPad). Statistical analysis of percentage of mice with tumors ≥ 1.0 cm3 versus time (days) was analyzed using a survival curve log-rank (mantel-cox) test in Prism5 (GraphPad).
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8

Dose-dependent Cytotoxicity of Vemurafenib and Temsirolimus

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Vemurafenib was purchased from ChemieTek (Indianapolis, IN, USA) and temsirolimus from Sigma-Aldrich (St. Louis, MO, USA). Both drugs were dissolved in dimethylsulfoxide (DMSO) and stock concentrations of 50 mM were stored at −20 °C in aliquots until use. Final concentrations were made in growth medium (0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 100 or 250 μM), before adding to the cell monolayers.
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9

Cell Viability Assay for Vemurafenib and Cytokine Treatment

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Cells were seeded in a 96-well flat bottom plate at a density of 2.0–2.5 x 103 cells/well and allowed to adhere for 24 hours in complete media. The following day, cells were treated with DMSO vehicle control and increasing concentrations of vemurafenib (PLX4032, Chemie Tek) in triplicate and cells were incubated for 72 hours. In all 8x8 matrix experiments, SB-3123 were treated with 0.1% BSA vehicle control, vemurafenib (0–16 uM), mouse IFNγ (0.8–51.2 ng ml−1), TNFα (0.08–5.12 ng ml−1), human IFNγ (0.4–100 ng ml−1), TNFα (0.04–10 ng ml−1) (PeproTech) and cells were incubated for 96 hours. Cells were analyzed using the CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer’s protocol (Promega).
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10

Synthesis and Reagents for Cell Assays

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G9 was synthesized by Cheminpharma (Branford, CT); Hemagglutinin-tagged ubiquitin vinyl methyl sulfone (HA-UbVS) was purchased from Boston Biochem; vemurafenib was purchased from Chemietek; PD 0325901 was purchased from Cayman Chemicals. Media components were obtained from Sigma and/or Invitrogen.
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