Cell culture and reagents. Four EGFR-mutant NSCLC cell lines in this study: HCCC827 (exon 19del E746-A750), PC-9 (exon 19del E746-A750), HCC4006 (exon 19del L747-E749), and HCC4011 (L858R). All the lines, except for HCC4011, were purchased from the American Type Culture Collection (Manassas, VA, USA). HCC4011 was kindly provided by Professor Adi F. Gazdar (University of Texas Southwestern Medical Center, Dallas, TX, USA). Five experimentally established EGFR-TKI-resistant cell lines, HCC827-GRS, resistant to Gefitinib by stepwise escalation methods; HCC827-GRH1, resistant to Gefitinib by highconcentration exposure methods; and PC-9-GRS, HCC4006-GRS, and HCC4011-GRS, which had acquired resistance to Gefitinib by stepwise escalation, as reported previously (31) (link). The resistance mechanisms of each drug-resistant cell are shown in Table I. All the cell lines were authenticated by short tandem repeat (STR) DNA analysis (Promega, Madison, WI, USA). All these cells were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum at 37˚C in a humidified incubator under 5% CO 2 gas. Gefitinib was purchased from InvivoGen (San Diego, CA, USA), and ganetespib was purchased from ChemScene (Monmouth Junction, NJ, USA).
DNA extraction. Genomic DNA was extracted from the cell lines with a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany).
DNA extraction. Genomic DNA was extracted from the cell lines with a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany).