Lentivirus-mediated Gene Knockdown

Lentivirus carrying shRNA sequence to the target gene was prepared using a three-plasmid transfection method [36 (link)] and used to infect cells. Cells with gene knockdown (SAE2^KD, EGFR^KD, FAK^KD, c-Met^KD or nAchRα7^KD) were selected using 1 μg/mL puromycin. The siRNA used for silencing gene expression was obtained from the National RNAi Core Facility (Institute of Molecular Biology/Genomic Research Centre, Academia Sinica, Taipei, Taiwan). Knockdown of cirRNA CCDC66 (cirRNA CCDC66^KD) was carried out by direct delivery of siRNAs (siJCT1 and siJCT2) [24 (link)] into H23 and H1975 cells using Ambion Silencer® siRNA transfection kit [10 (link)]. Reduction of cirRNA CCDC66 was determined by RT-PCR and gel electrophoresis.