Lentivirus particles

Lentivirus particles were purchased from Genechem. Lentivirus (LV)-enhanced green fluorescent protein (EGFP) was transduced into cells. The medium was replaced with fresh culture medium after 12 h. Cells were observed under a fluorescence microscope for transduction efficiency. Cells with stable expression were screened using puromycin.

Cell culture and construction of the stable cell line overexpressing Tspan9. Human GC SGC7901 cells were obtained from the Central Laboratory of the Affiliated Hospital of Qingdao University and were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37˚C under 95% humidity and 5% CO 2 content. Cells were harvested in the logarithmic growth phase for use in the experiments described below. The day before transfection, the SGC7901 cells were seeded into 6-well plates to ensure 60% confluency at the time of transfection. Then the cells were transfected with lentivirus particles (Genechem Co., Ltd., Shanghai, China) coding for Tspan9 or the negative control (NC), respectively, at a multiplicity of infection of 10. Six hours later, the medium was replaced, and cells were cultured for an additional 24 h and processed for further experiments. Transduction efficiency, as determined by electron microscopy (GFP, x200 magnification) ranged between 80 and 90%. The clones transfected with LV-Tspan9 were defined as the Tspan9 group, cells transfected with the negative control vectors were considered as the NC group, and the blank group included cells without transfection. Experiments were conducted with cell passages 3-20.