Lentivirus-Mediated Stable Gene Expression

For lentivirus-mediated stable introduction of sfGFP-DARPin-LA6 and NLS^SV40-sfCherry-NLS^Myc, we followed the methods described previously (Liu et al., 2021 (link)). Briefly, pVSV-G (PT3343-5; Clontech) and psPAX2 (plasmid #12260; Addgene; http://n2t.net/addgene:12260; RRID:Addgene_12260; a gift from Didier Trono), together with the pCDH vector (pCDH-NLS^SV40-sfCherry-NLS^Myc-Blast or pCDH-sfGFP-DARPin-LA6-Hygro) in a 1:3:4 weight ratio of each plasmid was transfected into ∼80% confluent 293T cells (CRL-3216; ATCC) using Lipofectamine 3000 following the manufacturer’s instructions for lentivirus production. One day after the transfection, the medium was replaced with fresh medium, which was harvested at 48 h after transfection. For virus infection, MEFs, C2C12, BJ-5ta, and MCF10A cells were incubated with the virus-containing culture supernatants with 4 µg/ml polybrene (Nacalai Tesque) for 24 h. Infected cells were selected by incubation in medium containing 200 µg/ml hygromycin B Gold or 3 µg/ml blasticidin S (InvivoGen) for 4 d, except that C2C12 cells were selected with 20 µg/ml blasticidin S for 4 d.