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4 protocols using trametinib

1

Compound Acquisition for Cell Experiments

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Crizotinib (PF-02341066) was obtained from ShangHai Biochempartner; cabozantinib (XL184), ceritinib (LDK378), lorlatinib (PF-06463922), trametinib (GSK1120212) from ActiveBiochem; foretinib from AdooQ Bioscience; brigatinib from Sellek; radicicol and rotenone from Cayman Chemical; 17-AAG (17-N-Allylamino-17-demethoxygeldanamycin) and GDC-0941 from LC Laboratories; TAE684 from ChemieTek; SB202190 from Cell Signaling Technology; SB239063 from Sigma; and cycloheximide and doxycycline hyclate from Nacalai Tesque. Each compound was prepared in dimethyl sulfoxide (DMSO), ethanol (WAKO) or distilled water for cell culture experiments.
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2

Synthesis and Characterization of Molecule14d

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DDP37368 (molecule14d) and its enantiomers DDP38003 (molecule15) has been synthesized as previously described68 (link). Details of other chemicals used in this study are Caspase-8 Inhibitor Z-IETD-FMK (R&D System, cat#FMK007), Caspase-3 Inhibitor Z-DEVD-FMK (R&D System, cat#FMK004), Pan Caspase Inhibitor Z-VAD-FMK (R&D System, cat#FMK001), m-TOR inhibitor rapamycin (LC Laboratories, R-5000 Rapamycin), IGF-1R/IR inhibitor OSI-906 (cat# S1091, Selleckchem.com), PI3K inhibitor Ly294002 (cat#9901, Cell Signaling Technology), ERK inhibitor Trametinib (GSK-1120212, cat#A-1258, Active Biochem), cycloheximide (Sigma, cat#C7698), actinomycin D((CAS 50-76-0); Santa Cruz Biotechnology, Inc., cat# sc200906) and TRAIL (ALX-201-073-C02, 3v Chimica Srl).
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3

Lung Cancer Cell Lines Characterization

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The lung cancer cell lines A549, NCI-H358, NCI-H1792, NCI-H23, SW900 and SW1573 were purchased from the American Type Culture Collection. SK-LU-1, Calu-1, Calu-6 and NCI-H460 were provided by Takashi Takahashi (Nagoya University, Japan). RERF-LC-AD2, LU-65 and LU-99 cells were obtained from the Japanese Cell Research Bank (Osaka, Japan). HCC2108 cells were obtained from Korean Cell Line Bank (Seoul, South Korea). The NCI-H1573 and NCI-H2030 cells were provided from Massachusetts General Hospital Cancer Center. Cells were cultured in RPMI1640 (Invitrogen) with 10% FBS. Characteristic of cell lines used in this study were summarized in Supplementary Table S4. Cells were obtained between 2012 and 2016. Experiments using A549, SW900, LU-65 and HCC2108 cells were done within 6 months from the acquisition of these cells from cell banks. All other cell lines were tested and authenticated by short tandem repeat (STR) analysis with GenePrint 10 System (Promega) by the Japanese Cell Research Bank at the time of submission. Cells were regularly screened for Mycoplasma using a MycoAlert Mycoplasma Detection Kit (Lonza). NVP-BGJ398, GDC-0941, ABT-263, selumetinib, afatinib, and trametinib were obtained from Active Biochem. Compounds were dissolved in DMSO to a final concentration of 10 mmol/l and stored at −20°C when not in use.
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4

Inhibiting Cancer Cell Metabolism

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AURKA inhibitor alisertib (MLN8237, number S1133) and CDK4/6 inhibitor palbociclib (number S1116) were purchased from Selleck Chemicals (Munich, Germany). MAPK/ extracellular signalÀregulated kinase kinase 1/2 inhibitor trametinib (GSK1120212, catalog number A-1258) and BRAF inhibitor dabrafenib (catalog number A-1220) were purchased from Active Biochem (Hong Kong, China). Inhibitor of mitochondrial beta-oxidation (inhibitor of the carnitine palmitoyltransferase CPT1) etomoxir (HY-50202/cs-3271) was purchased from MedChemExpress (Monmouth Junction, NJ). For in vitro studies, compounds were resuspended in DMSO at 10 mM (alisertib, trametinib), 20 mM (dabrafenib), and 80 mM (etomoxir) stock concentration.
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