Lentivirus-mediated overexpression

Slug-overexpressing SK-OV-3 and A2780 EOC cell lines were established via lentivirus transfection. A lentiviral vector encoding slug (LV-slug) and a negative control vector (LV-vector) were purchased from OBiO Technology (Shanghai) Corp., Ltd., and carried an enhanced green fluorescent protein reporter gene, EGFP. The lentiviral vector used for overexpression was named pSLenti-CMV-EGFP-3×FLAG-PGK-Puro-WPRE according to the manufacturer's introduction. For slug exogenous overexpression, lentivirus containing LV-slug or the LV-vector were transfected into SK-OV-3 and A2780 cells using Polybrene (5.0 µg/ml) from OBiO Technology according to the manufacturer's protocol. Briefly, cells were seeded into a 6-well plate (1×10⁵ cells/well) for adherence. Then, the cells were transfected with lentivirus vectors (LV-slug or LV-vector) at a multiplicity of infection of 30, in the presence of 5 µg/ml Polybrene at 37°C. After 72 h of transduction, medium containing puromycin (0.2 mg/ml) was added to select stably transduced cells, and stable cell lines were screened for 3 weeks. Slug upregulation efficiency was assessed by western blotting.