Polyjet

To generate HEK 293 cell line harboring SuperTopFlash (STF) reporter gene, 125 ng of the STF construct (Kelei Biological Technology Co., Ltd., Shanghai, China) and 125 ng of pSV2neo (Kelei Biological Technology Co., Ltd., Shanghai, China) providing a neomarker for dominant selections screening out by 600 μg/ml of G418 (Invitrogen Corporation, Carlsbad, California, USA) were stably transfected into HEK 293 cells in a 48-well plate using PolyJet (Invitrogen Corporation, Carlsbad, California, USA) [16 (link)]. The HEK 293 cell lines harboring STF reporter gene were cotransfected with 200 ng of Norrin (wild type or mutant), 200 ng of FZD4 (wild type or mutant), 200 ng of LRP5, and 100 ng of pSV-β-gal (Kelei Biological Technology Co., Ltd., Shanghai, China) in a 48-well plate using PolyJet (Invitrogen Corporation, Carlsbad, California, USA). Forty-eight hours after transfection, we washed the cells twice with 1x PBS and subjected to luciferase assay using Dual Luciferase assay reagent (Promega, USA).

For the virus infection, the expression vectors were transfected together with package vectors (psPAX2 and pMD2.G) into HEK293T cells by Lipofectamine 2000 (Thermo Fisher Scientific, Cat. No. 11668) or by PolyJet (SignaGen laboratories, Cat. No. SL100688). After 48 h, the supernatants of the medium were collected and filtered with 0.45 μm filter. The supernatant containing the virus was stored at 4 °C for cell infection. The breast cancer cells were cultured in fresh media and subsequently infected with lentivirus overnight together with Polybrene (Sigma, Cat. No. H9268). For cell transfection, Lipofectamine 2000 (Invitrogen) or PolyJet was utilized according to the protocol.