Mission lentiviral packaging mix kit

The recombinant lentiviral plasmids, the packaging vector pGag/Pol, pRev, and the envelope vector pVSV-G were co-transfected into HEK293T cells using MISSION Lentiviral Packaging Mix kit (Sigma-aldrich). The mixture, including 20 μL Packing Mix (PVM), 12 μL PEI, and 400 μL serum-free DMEM and 20 μg purified plasmid DNA, was incubated at room temperature for 15 min and transferred to HEK293T cells at 70–80% confluence. The medium was replaced with fresh complete medium after 16 h incubation, and the cells cultured for 48 h. The supernatant were collected, purified, and concentrated with a Centricon Plus-20 centrifugal ultrafiltration device (Millipore).

pLKO.1-puromycin vectors containing control shRNA (gift from Dr. David Sabatini^{99 (link)}, Addgene plasmid Cat# 1864) or 4E-BP1 shRNA (gift from Dr. Tommy Alain^{100 (link)}, Sigma Cat# TRCN0000040203) were packaged using MISSION® Lentiviral Packaging Mix kit (Sigma Aldrich Cat# SHP001) according to manufacturer’s protocol. WT or 3SA MCF10A cells were infected with the lentivirus particles and selected using puromycin. Stable 4E-BP1 knockdown was confirmed using western blots (Fig. 7a).