Helper 2

The package of lentivirus was used 3rd generation system. The GV115 (pGCSIL-GFP; Shanghai GeneChem Co., Ltd.) lentivirus was used for constructing GBAS-knockdown vectors. The short hairpin RNA (shRNA/sh) sequence targeting GBAS used was as follows: Forward, 5′-CCGGCCATGTGAAAGTCCTGTTGAACTCGAGTTCAACAGGACTTTCACATGGTTTTTG-3′ and reverse, 5′-AATTCAAAAACCATGTGAAAGTCCTGTTGAACTCGAGTTCAACAGGACTTTCACATGG-3′. After cloning the shGBAS or shCtrl sequence into the GV115 vectors, the lentivirus construction system, including the GV115-GBAS vector (20 µg), and Helper 1.0 (15 µg; Shanghai GeneChem Co., Ltd.) and Helper 2.0 (10 µg; Shanghai GeneChem Co., Ltd.) packaging vectors, were co-transfected into 293T cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following 72 h of transfection, the freshly harvested virus supernatants were collected by ultracentrifugation (25,000 × g at 4°C for 2 h) and subsequently stored at −80°C. The multiplicity of infection for EC cell transfection was 20. After 72 h, the transfection efficiency of GBAS knockdown (shGBAS) was determined using reverse transcription-quantitative PCR (RT-qPCR) and western blotting.

To generate A549 and H1299 cell lines stably overexpressing QKI‐5, a ~ 1‐kb coding sequence (GenBank Accession number: NM_006775) of QKI‐5 was subcloned into a GV358 lentiviral expression vector (GeneChem Inc., Shanghai, China) containing a C‐terminal 3× Flag tag using endonucleases AgeI/BamhI (Fermentas, Glen Burnie, MD, USA). Primers used for amplifying the coding sequence of QKI‐5 were listed in Appendix Table S5. The empty lentivirus vector was served as a negative control. Subsequently, the QKI‐5 expression construct or empty vector was co‐transfected with packaging plasmids Helper 1.0 and Helper 2.0 (GeneChem Inc.) into HEK 293T cells using Lipofectamine 3000 (Invitrogen). At 48 h post‐incubation in DMEM with 10% FBS, the packaged lentiviruses were collected and used to infect A549 and H1299 cells for 72 h. Finally, Flag‐tagged QKI‐5‐overexpressing A549 and H1299 stable cells were selected with 2 μg/ml puromycin (Solarbio Lifesciences, Beijing, China).