Plenti puro

All third-generation lentiviral plasmids were obtained from Addgene (Watertown, MA, USA). The envelope (pMD2.G; Addgene plasmid #12259; http://n2t.net/addgene:12259;RRID: Addgene_12259) and packaging plasmids (pRSV-Rev; Addgene plasmid #12253; http://n2t.net/addgene:12253;RRID: Addgene_12253 and pMDLg/pRRE; Addgene plasmid # 12251, http://n2t.net/addgene:12251;RRID: Addgene_12251) were gifts from Dr Tronoa (32 (link)). The transfer plasmid was a gift from Ie-Ming Shih (33 (link)) (pLenti-puro; Addgene plasmid #39481; http://n2t.net/addgene:39481;RRID: Addgene_39481). The pLenti-puro FLAG-tagged FOXL2 was generated by PCR using pcDNA3 FLAG-tagged FOXL2 as the template and the primers FLAG-FOXL2-F and FLAG-FOXL2-R. The PCR products were digested with BamHI and XbaI (Takara Bio) and ligated into the pLenti-puro vector (Addgene). 293T cells were used for lentivirus production by transfection of 12 μg of pMDLg/pRRE and pRSV-Rev, 8 μg of pMD2.G and 16 μg of pLenti-puro empty vector or pLenti-puro-FLAG-FOXL2 for 48 h. Viral supernatant was collected for KGN cell infection in the presence of 10 μg/ml polybrene (H9268, Sigma-Aldrich) (0.5 ml of lentivirus to 5 × 10⁴ cells in 1.5 ml of medium). After 48 h of infection, cells were selected with 2 μg/ml puromycin (P8833, Sigma-Aldrich) for 30 days.