Generation of PLK4-inducible Stable Cell Lines

To generate PLK4‐inducible stable cell lines from OVCAR8 and SKOV3 cells, we used a doxycycline‐inducible PLK4 lentiviral expression system (Holland et al, 2012 (link)). Viruses were produced in HEK293T cells, co‐transfected with two other vector plasmids using lipofectamine 2000: a vesicular stomatitis virus envelope expression plasmid (Vsvg) and a second‐generation packaging plasmid (pPax2). Viral particles were then used to infect OVCAR8 and SKOV3 cell lines for 24 h. Infected cells were selected using bleomycin 50 μg/ml (Santa cruz Biotechnology #sc200134A) for 15 days. Newly generated stable cell lines iOVCAR8 and iSKOV3 were then expanded in DMEM/F12 media supplemented with 10% tetracycline‐free fetal bovine serum (FBS, Dutscher #S181T), streptomycin (100 μg/ml,), and penicillin (100 U/ml). To induce PLK4 overexpression, cells were treated with doxycycline (1 μg/ml) for 96 h.