Plenti6.3 v5 dest gateway vector

Lentiviral clones containing two shRNA sequences against human MIF and a control sequence (GE Dharmacon), lentivirus packaging plasmid psPAX2, envelope plasmid VSV-G, and transfection reagent FuGENE6 (Promega) were used to produce viral particles from HEK293T cells. Lentivirus was added to U87-MG and LN229 cells in DMEM media supplemented with 10% FBS and polybrene (Santa Cruz Biotechnology) per manufacturer’s protocol. To overexpress MIF, human MIF cDNA was generated as a PCR product (primers in Supplementary Table S2) from a plasmid containing MIF cDNA (Origene) in the pENTR/D-TOPO Cloning Kit (Invitrogen), sequenced, and cloned into the pLenti6.3/V5-DEST Gateway Vector (Life Technologies, Carlsbad, CA, USA). Cultured U87-Bev^R cells were transduced with empty or MIF-containing vector, from which U87-Bev^R/EV (empty vector) along with U87-Bev^R/MIF1 and U87-Bev^R/MIF2, two clones overexpressing MIF relative to U87-Bev^R, were selected, with resulting cells GFP⁺ due to the vector GFP gene.