Plenti pgk lifeact gfp w

HEK293T high performance cells (ATCC) were transfected with pLenti.PGK.LifeAct-GFP.W (Addgene plasmid 51010). The cell-free supernatant was collected after 48 h and the virus titer was enriched via ultracentrifugation. Primary human basal mammary epithelial cells were transduced and after 24 h, cells were trypsinized and seeded into floating collagen gels.

cDNA for pLenti CMV GFP Puro (658-5) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid #17448^{76 (link)}). Portions of paxillin-pEGFP (a gift from Rick Horwitz, Addgene plasmid # 15233) or mRuby-Paxillin-22 (a gift from Michael Davidson, Addgene plasmid #55877) were subcloned into a modified version of the pRRLSIN.cPPT.PGK-GFP.WPRE (a gift from Didier Trono, Addgene plasmid # 12252) by restriction enzyme cloning. pLenti.PGK.LifeAct-GFP.W was a gift from Rusty Lansford (Addgene plasmid # 51010). To generate lentivirus, plasmids were co-transfected with pCMV-VSVG (a gift from Bob Weinberg, Addgene plasmid #8454), pMDLg/pRRE, and pRSV-REV (gifts from Didier Trono, Addgene plasmid #12251 and #12253^{77 (link),78 (link)}) in 293T cells using the calcium phosphate precipitation method⁷⁹ . Viral supernatants were collected after 48 h, concentrated with PEG-it^TM (System Biosciences, Palo Alto, CA) following the manufacturer’s protocol, filtered through a 0.45 μm filter (ThermoFisher Scientific Nalgene, Waltham, MA), and stored at −80 °C. Viral titer was determined by serial dilution and infection of NIH3T3s in the presence of 10 μg mL⁻¹ polybrene (Santa Cruz Biotechnology, Dallas, TX). Titers yielding maximal expression without cell death or detectable impact on cell proliferation or morphology were selected for studies.