Generation of stable cell lines overexpressing CPNE1

To generate NSCLC cells in which CPNE1 is stably overexpressed, a 1626-bp fragment of the CPNE1 coding sequence was synthesized (Genewiz, Suzhou, China) and subcloned into a pLVX-IRES-Neo vector using the endonucleases EcoRI and XbaI for expression via a Lenti-X lentiviral expression system (Clontech, Mountain View, CA, USA). The CPNE1 expression construct was co-transfected with packaging plasmids into human embryonic kidney 293 T cells using Lipofectamine 2000 (Invitrogen). The empty vector served as a negative control. Human embryonic kidney 293 T cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS at 37 °C in a humidified 5% CO₂ incubator for 48 h. After the incubation, the packaged lentiviruses were collected and used to infect A549 and H1299 cells. After 2 days, stable cells were selected with 400 μg/ml of G418 (Amresco, Solon, OH, USA).