Penicillin streptomycin amphotericin b

Metastatic murine melanoma cells (B16F10) are incubated in Dulbecco’s modified Eagle’s medium nutrient mixture F12 (DMEM/F12, Gibco) containing 10% fetal bovine serum (FBS, Bio-Channel) and 1% antibiotic (penicillin/streptomycin/amphotericin B, Beyotime) solution in a humidified atmosphere with 5% CO₂ at 37°C.

Two RAS‐driven LUAD cell lines (A549 and NCI‐H1299) were purchased from the Chinese Academy of Science Cell Bank (https://www.cellbank.org.cn/). The cell lines were authenticated by short tandem repeat (STR) profiling before use. Cells were cultured in the DMEM (High Glucose), supplemented with 10% fetal calf serum (Every Green, Zhejiang Tianhang Biotechnology), as well as penicillin/streptomycin/amphotericin B (Beyotime Biotechnology). Cells were cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37°C.
They were transfected by lentivirus containing green fluorescent protein (GFP), and untransfected cells were filtered out by flow cytometry. Then GFP‐included cells were transfected with siRNAs at a 100 nM final concentration using the Lipofectamine8000 (Beyotime) and the Opti‐MEM (Thermo Fisher Scientific). siRNAs were designed by and purchased from the Guangzhou RiboBio Co, China. We used two different siRNAs separately targeting each gene (IRX2, SPINK13, and CAPN8) and two different siRNAs without targeting the human genome as controls. The targeting sequences of each siRNA can be accessed in the Table S2. The efficiency was assessed using quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis after transfection. Primers used can be obtained in the Table S3.