Lentiviral shrna construct targeting

Lentiviral shRNA construct targeting FOXP1 was purchased from Sigma–Aldrich (Louis, MO). For generation of lentiviral particles, 293FT cells were transfected with the 6.67 μg targeted viral plasmid and lentiviral packaging mix; 5 μg of VSV-G and 3.33 μg of Δ8.9 using 15 μL Lipofectamine/Lipofectamine Plus reagent. For retroviral overexpression of FOXP1, FOXP1 cDNA was subcloned into BamHI and XhoI sites of the pMXs IRES GFP retroviral vector (#RTV-013; Cell Biolabs, Inc.) and then transfected with the 5 μg targeted viral plasmid, 5 μg of VSV and 5 μg of gag/pol into 293FT cells by using Lipofectamine/Lipofectamine Plus reagent. The culture supernatants containing lentivirus or retrovirus were harvested and concentrated using the Lenti-X Concentrator (#631231; Clontech) and Retro-X Concentrator (#631445; Clontech) at 48 h after transfection. For lentiviral or retroviral transduction, A2780 cells were infected with shRNA-bearing lentivirus or pMXs-RNA-bearing retrovirus in the presence of 5 μg/mL polybrene (Sigma-Aldrich). FOXP1-silenced or –overexpressed cells were selected for 10 days with 1μg/mL puromycin and then maintained in RPMI1640 supplemented with 10% FBS and 1 μg/mL puromycin.