Fig. 4

The role of PRRs in IFIT induction and hBMEC barrier maintenance. A–C hBMECs were infected with JEV P3 for different time points, and the mRNA and protein levels of TLR3 (A), RIG-I (B), and MDA5 (C) were measured. D, E PRR-knockdown hBMECs were infected with JEV P3, and the expression of TLR3, RIG-I, and MDA5 was assessed by RT-qPCR (D) and Western blotting (E). F PRR-knockdown hBMECs were serum starved after reaching confluence, and cell viability was measured. G, H Expression of IFITs was assessed by RT-qPCR (G) and Western blotting (H) in PRR-knockdown hBMECs. I Confluent monolayers of hBMECs in the upper chamber of the transwell plate were infected with JEV P3, and the TEER (upper graph) was measured at the indicated times. J hBMECs were seeded onto the upper chamber, cultured astrocytes infected with JEV P3 or heated-JEV P3 were placed in the lower compartment of the transwell plate, and the TEER (upper graph) was measured at the indicated times. K The culture supernatants from either mock-infected or JEV-infected astrocytes were collected and mixed with an equal volume of fresh medium (Mock-CM/JEV-CM) and added to confluent hBMECs in the upper chamber of the Transwell plate. Then, the TEER was measured at the indicated times. L, M PRR-knockdown hBMECs were seeded onto the upper chamber of the Transwell plate until confluent. The TEER (upper graph) was measured without treatment (L) or with mock-CM/JEV-CM treatment (M) at the indicated times. Data are represented as the mean values ± SEMs from three independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant