Lentiviral Inducible Expression and CRISPR-Cas9 Editing

For doxycycline-inducible lentiviral expression of a constitutively nuclear human FOXO1 (FOXO1^A3), FLAG-tagged FOXO1^A3 was cloned into pLVX-TetOne-Puro (Clontech). For CRISPR–Cas9 genome editing, gene-specific gRNA sequences (Supplementary Table 4) were cloned into a plentiCRISPRv2 plasmid co-expressing FLAG-tagged Cas9 nuclease and a puromycin-selection marker (Addgene, 52961). The FUCCI lentiviral reporters mCherry-hCdt1(30/120)-pCSII-EF and mVenus-hGeminin(1/110)-pCSII-EF^{56 (link)} were obtained from the Laboratory for Cell Function Dynamics, CBS, RIKEN, Japan.
Lentivirus production was performed by co-transfection of HEK293FT cells with pMD2.G (Addgene, 12259), psPAX2 (Addgene, 12260) and transfer plasmids, accordingly. Transfections were carried out using Lipofectamine 2000 transfection reagent (Life Technologies) as previously described^{57 (link)}. Viruses were collected 48 and 72 h after transfection, filtered through a 0.45-μm filter and incubated with HUVECs for 16 h in the presence of 8 µg ml⁻¹ polybrene (Santa Cruz). After transduction, cells were expanded for 48 h and selected with EBM containing 1 µg ml⁻¹ puromycin (InvivoGen, ant-pr-1).