Unc569

Phorbol 12-myristate 13-acetate (PMA), polyinosinic acid potassium salt (poly I), cytochalasin D, bafilomycin A1, BMS345541, and adenosine 5′-triphosphate disodium salt hydrate (ATP) were purchased from Sigma Aldrich (St. Louis, MO, USA). zYVAD-fmk and UNC569 were purchased from Merck (Darmstadt, Germany).

PC-9 cells (EGFR Ex19 del E746_A750) were purchased from the European Collection of Cell Cultures in 2014. RPC-9 cells (Gefitinib-resistant; EGFR Ex19 del E746_A750 and Ex20 T790M) were established from a parental PC-9 cell line in our laboratory^{18 (link)}. HCC827 cells (EGFR Ex19 del E746_A750)^{5 (link)} and PC-9/BRc1 cells (afatinib-resistant; EGFR Ex19 del E746_A750 and Ex20 T790M)^{19 (link)} were kindly provided by Dr. William Pao (Vanderbilt University, Nashville, TN, USA). Cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin in a tissue culture incubator at 37 °C under 5% CO₂.
Naquotinib was provided by Astellas Pharma Inc. (Tokyo, Japan) under a material transfer agreement. Gefitinib, afatinib, osimertinib, crizotinib, SGX-523, selumetinib, and trametinib were purchased from Selleck Chemicals (Houston, TX, USA). UNC569 was purchased from Merck Millipore (Billerica, MD, USA). All compounds were dissolved in dimethyl sulfoxide for in vitro studies.
Growth inhibition was measured using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay^{20 (link)}. Briefly, cells were plated onto 96-well plates at a density of 2,000–3,000 per well and continuously exposed to each drug for 96 h.