Pgk h2bmcherry

pMXs-c-Myc was a gift from Shinya Yamanaka (Addgene plasmid #13375); pBABE-puro-RAS V12 was a gift from Bob Weinberg (Addgene plasmid #1768); MSCV-p53DD-iGFP was a gift from Wechsel-Reya lab; PGK-H2BmCherry was a gift from Mark Mercola (Addgene plasmid # 21217); PIK3CA^H1047R was subcloned from pBabe-puro-HA-PIK3CA^H1047R, a gift from Jean Zhao (Addgene plasmid # 12524), into PGK-H2BmCherry; 7xTcf-eGFP//SV40-PuroR (7TGP) was a gift from Roel Nusse (Addgene plasmid #24305); pTween-Luc-NOeGFP was a gift from Stassi lab. Inducible IPTG-driven shRNAs for Myc and constitutive shRNAs for Myz-1 were purchased from Sigma-Aldrich.

For validation of EPIKOL screen candidate hits, dual color competition assays were performed. Cas9-stable cells were transduced with either PGK-H2BmCherry (Addgene #21217) or PGK-H2BeGFP (Addgene #21210) viruses at high MOI ~5 to make sure every cell was fluorescently labeled. 50,000 cells were seeded in 12 well-plates, mCherry+ cells were transduced with LentiGuide-NT1 viruses, while eGFP+ cells were transduced with viruses carrying sgRNA-X for selected genes. For each gene, two different sgRNAs were used (Supplementary Table 2). After 16 h, viral media were changed with fresh media and next day puromycin selection was started. After 3 days of puromycin selection, mCherry+ and eGFP+ cells were mixed in a 1:1 ratio and re-seeded into 24-well plates in triplicates. One day after seeding, Day0 measurements were taken by acquiring 3 × 3 images with a 4× objective in Cytation5 (BioTek, USA). Cells were incubated for the subsequent 16 days, and images were taken at Day4, Day8, Day12, and Day16. Number of mCherry+ and eGFP+ cells were counted from images using Gen5 software (BioTek, USA) and each measurement was normalized to Day0 to determine the percentage of eGFP+ cells.

NPC lines genetically modified for the expression of H2B-mcherry fusion protein were generated and used in the long-term live-cell imaging experiments. Lentiviral vectors were generated by transient transfection of HEK293FT cells with psPAX2, pMD2G, and PGK-H2BmCherry (Addgene plasmids: #12260, #21217, #12259)35 (link). NPCs were transduced with H2B-mCherry lentiviral vectors, expanded and imaged using the Operetta High Content Imaging System (PerkinElmer). The cells were mock- or ZIKV-infected and imaged from 24 to 72 h after infection. The cells were maintained in a chamber with controlled temperature (37 °C) and atmosphere (5% CO₂). Images were captured at multiple points every 10 min. At least 500 mitoses were analyzed for each condition and were assessed for the presence of chromosome laggards, multipolarity and apoptosis induction during mitosis or interphase.