Filter units

A full-length human nephrin coding sequence was generated by PCR from a pCMV-SC-CF plasmid (a kind gift from Puneet Garg at University of Michigan School of Medicine, Ann Arbor, MI, USA). Nephrin was cloned into a pGEM-T vector (Promega) and subcloned into a CSII-CMV-MCS-IRES2-Venus vector using the NheI and NotI sites. The lentivirus vectors were kindly provided by Dr Hiroyuki Miyoshi (Riken BioResource Center, Tsukuba, Japan). All of the lentiviral vectors were generated by transient transfection of 293T cells using the calcium phosphate precipitation method. Briefly, 293T cells were seeded into 100 mm dishes with DMEM medium containing 10% FBS. The mixture of the main plasmid and the packaging plasmids was transfected into each dish by calcium phosphate precipitation. The supernatants collected at 48 and 72 h were concentrated and purified by ultracentrifugation with filter units (Millipore, Schwalbach, Germany), and then the lentivirus was titrated onto HT1080 cells. BeWo cells were infected with nephrin or empty lentivirus and passaged three times to purity by flow cytometry to ensure a transfection rate of more than 95%.

Human osteosarcoma U2OS and colon cancer HCT116 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Carlo Erba), supplemented with 1% L-glutamine and 10% FBS (fetal bovine serum). U2OS cells were seeded in 35 mm dishes at a concentration of 100000 cell/ml. Filter Units (Millipore), and imidazole was finally removed by buffer exchange with PBS pH 8.0 with Amicon Ultra-15 Centrifugal Filter Units.