Lentivirus-based small hairpin RNAs (shRNA) were from Sigma-Aldrich. psPAX2 and pMD2.G combined with specific shRNA were co-transfected into 293 T cells. Replace the culture medium with fresh DMEM with 10% FBS 24 h after transfection. 48 h later, the virus containing medium was collected and added to cancer cells with polybrene (12 μg/mL). Puromycin selection was performed at concentration ranges between 3 and 5 μg/mL 48 h after infection. The sequence information of shRNA is provided in Supplementary Table 2 .
The pTsin lentiviral expression vector was used to generate lentiviral plasmids for Tsin-NFAT1, which could stably overexpress NFAT1 in cells as previously reported [20 (link)]. Lipofectamine 2000 was used to transfect 293 T cells with the pTsin expression plasmid and viral packaging plasmids (pHR’ CMVδ 9.8 and pVSV-G). Twenty-four hours after transfection, the medium was replaced with fresh DMEM, containing 10% FBS and 1 mM of sodium pyruvate. Next, 48 h post transfection, the virus culture medium was collected and added to renal cancer cells supplemented with 12 μg/ml of polybrene. Twenty-four hours after infection, the infected cells were selected with 10 μg/ml of puromycin.
The pTsin lentiviral expression vector was used to generate lentiviral plasmids for Tsin-NFAT1, which could stably overexpress NFAT1 in cells as previously reported [20 (link)]. Lipofectamine 2000 was used to transfect 293 T cells with the pTsin expression plasmid and viral packaging plasmids (pHR’ CMVδ 9.8 and pVSV-G). Twenty-four hours after transfection, the medium was replaced with fresh DMEM, containing 10% FBS and 1 mM of sodium pyruvate. Next, 48 h post transfection, the virus culture medium was collected and added to renal cancer cells supplemented with 12 μg/ml of polybrene. Twenty-four hours after infection, the infected cells were selected with 10 μg/ml of puromycin.