Fig. 5

The role of IFNs in IFIT induction and hBMEC barrier maintenance. hBMECs were infected with JEV P3, the cells/cell culture supernatants were harvested at 0, 12, 36, and 72 hpi, and the expression of IFN-β/IFN-λ1 at the mRNA and protein levels was determined by utilizing RT-qPCR (A, B) and ELISA (C, D). E, F hBMECs were treated with 10 ng/ml rhIFN-β (E) or 100 ng/ml rhIFN-λ1 protein (F) for different time points, and IFIT expression was measured by Western blotting. G, H HEK-293T cells were transfected with either empty vector plasmid (vector) or IFN-β (Vector-IFN-β)/IFN-λ1 (Vector-IFN-λ1) expression plasmid as indicated, and IFIT expression was measured by performing RT-qPCR (G) and Western blotting (H). I, J Vero cells were treated with poly I: C or rhIFN-β/rhIFN-λ1 protein as indicated, and IFIT expression was measured by performing RT-qPCR (I) and Western blotting (J). K HEK-293T cells were transfected with either empty vector plasmid (vector) or plasmids expressing IFIT1 (Vector-IFIT1), IFIT2 (Vector-IFIT2), IFIT3 (Vector-IFIT3), and IFIT5 (Vector-IFIT5) as indicated, and IFN-β and IFN-λ1 mRNA expression was measured by performing RT-qPCR. (L) Dual-luciferase reporter assays detected IRF1 binding to the IFN-β promotor in IFIT-overexpressing HEK-293T cells. HEK-293T cells were transfected with vector-IFIT1, vector-IFIT2, vector-IFIT3, vector-IFIT5, or empty vector plasmid to generate overexpressed cell lines. Simultaneously, empty vector plasmid or vector-IRF1 and IFN-β-Luc alone with pRL-TK plasmids were cotransfected into corresponding overexpressed cell lines. M hBMECs were cultured in the upper chamber of the transwell plate and pretreated with rhIFN-β and rhIFN-λ1 protein, followed by treatment with mock-CM or JEV-CM, and then the TEER (upper graph) was determined at the indicated times. Data are represented as the mean values ± SEMs from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant