Pc 9 cells

Human non-small-cell lung cancer (NSCLC) A549 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Human NSCLC PC-9 cells were procured from Immuno-Biological Laboratories (Gunma, Japan). A549 cells were propagated in low-glucose DMEM, and the PC-9 cells were propagated in the RPMI-1640 medium. Both media contained 1% P/S and 10% inactivated FBS. A549 and PC-9 cells were maintained at 37 °C in 5% CO₂.

Four EGFR‐mutant lung adenocarcinoma cell lines were used in this study. HCC827 cells (with a deletion in exon 19) were purchased from the American Type Culture Collection and PC‐9 cells (with a deletion in exon 19) were obtained from Immuno‐Biological Laboratories. These cells were obtained from 2010 to 2016. Osimertinib‐resistant HCC827 (HCC827‐OR) and osimertinib‐resistant PC‐9 (PC‐9‐OR) cell lines were established using a stepwise method, as previously described.¹⁷ Cells were amplified and frozen, and one aliquot of each was thawed for this research. All cells were routinely screened for the absence of mycoplasma and cultured in RMPI 1640 medium (FUJIFILM Wako Pure Chemical Co.) with 10% heat‐inactivated fetal bovine serum (FBS) (BioWest) and 1% penicillin and streptomycin (FUJIFILM Wako Pure Chemical Co) at 37°C in a 5% CO₂ incubator. Falcon Cell Culture Inserts (Corning Inc.) with a transparent polyethylene terephthalate membrane (pore size: 0.4 μm) were used for co‐culture. HCC827 and PC‐9 cells were plated into six‐well plates (2 × 10⁵ cells/well), the device was placed on the six‐well plates, and equal numbers of their respective resistant cells were plated into the device. After 72 h, the device was removed and HCC827 and PC‐9 cells were used for protein extraction.