Lentiviral Transduction of Murine Lung Cells

LLC, E0771, and MMTV-PyMT parental cells were maintained as previously described^{4 ,24 (link),46 (link)}. GFP and luciferase were cloned into the pCDH-puro plasmid. The lentiviral vector pLX311-luciferase was a gift from William Hahn (Addgene plasmid #117735). Parental cells were transduced with pCDH-GFP-luc or pLX311-luc. GFP⁺ cells were enriched using cell sorting. Murine pulmonary microvascular endothelial cells (MPMECs) were isolated from 8 to 16-week-old Rptor^fl/fl or Tsc2^fl/fl mice and maintained in EGM-2 medium (Lonza), as previously described^{71 (link)–75 (link)}. For adenoviral Cre expression, MPMECs were seeded onto tissue culture plates coated with 0.1% gelatin at 70-80% confluency. Cells were infected with 10⁷ PFU ml⁻¹ of Ad-CMV-iCre (Vector Biolabs, #1045) for 16-48 hours, as indicated. Ad-CMV-b-Gal (Vector Biolabs #1080) or Ad-CMV-Null (Vector Biolabs #1300) were used as control vectors as indicated. Cells were harvested 48-72 hours after transduction.