Lentiviral Transduction of Cancer Cell Lines

HEK293T cells were cultured until 60% confluence in 6 cm plates and cotransfected with 2 μg of the appropriate plasmids and 2.5 μg of helper plasmids (1.5 μg psPAX2 and 1 μg pmD2.G) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The supernatants containing viral particles were collected 48 h and 72 h later and filtered through 0.45 μm polyvinylidene fluoride (PVDF) membranes. MDA-MB-231, MCF-7, and 4T1 cells were transduced with viral supernatants containing 5 μg/mL polybrene, and the medium was replaced 48 h later. The stably transduced cells were selected by culturing in the presence of 2 μg/mL puromycin for at least 7 days. Subsequently, the cells were harvested for Western blotting.