Lentiviral Transduction for Stable Expression

Transient transfection with plasmids was performed using GenJet DNA transfection reagent (SignaGen Laboratories, SL100489) according to the manufacturer’s instruction. Lentiviral transduction was performed as before for stable expression of the mitophagy reporter mito-mCE, shRNAs, and sgRNAs [45 (link)]. In brief, to produce infectious lentiviruses, 293T cells were co-transfected with a lentiviral transfer vector together with the packaging plasmid psPAX2 (a gift from Didier Trono, Addgene plasmid #12260) and the vesicular stomatitis virus G protein expression plasmid pVGV-G at a ratio of 5:4:1 for 2 days. Transduction units (TUs) of 60x concentrated lentiviruses were determined in 293T cells in the presence of appropriate antibiotics to select transduced cells. Target cells were transduced with lentiviruses at a single dose of 10 TUs in the presence of 10 μg/ml polybrene for 6 h. Stably transduced cells were selected for 2–3 weeks in the presence of appropriate antibiotics.