EGFR-mutant Lung Cancer Cell Culture

The human lung cancer cell line PC9 harboring del E746-A750 activating mutation in EGFR was maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and incubated in a humidified atmosphere of 5% CO₂ at 37°C. The PC9 cells were kindly provided by Dr. Mayumi Ono (Kyushu University, Fukuoka, Japan) (13 (link), 19 (link), 23 (link)). Cells were routinely confirmed to be free of mycoplasma contamination using mycosensor QPCR Assay kits (Agilent Technologies). Afatinib, lapatinib, foretinib, gefitinib, and dasatinib were purchased from Selleck (Houston, USA). PD173074, cisplatin, paclitaxel and axitinib were from Sigma Aldrich (St. Louis, MO). The construction of pcDNA3-Twist has previously been described (24 (link)). The small interfering RNAs (siRNA) corresponding to FGFR1, Twist1, ZEB1, Snail, and Slug, mRNA and a non-specific siRNA (control) were purchased from Nippon Gene (Tokyo, Japan). Cells were transfected with siRNA duplexes using Lipofectamine RNAiMAX and Opti-MEM (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations.