TLR3 activation induced IFN-β and IFN-λ expression. (A) hCMEC/D3 cells were stimulated with 0.1 µg/mL PolyI:C-Rhodamine for 16 hours and cultured for 48 hours poststimulation. Cells were fixed and stained with antibody to ZO-1 tight junction protein (mouse; 1:100; green). After nuclear counterstaining with Hoechst dyes, cells were observed under a fluorescence microscope. White arrows indicate the internalization of PolyI:C (magnification ×100). (B) Effect of PolyI:C on IFN expression of brain ECs. RNA extracted from the cells was subjected to real-time polymerase chain reaction (RT-PCR) for IFNs as indicated. (C,D) Dose-dependent effect of PolyI:C on IFN induction of hCMEC/D3 cells at (C) mRNA and (D) protein levels. Data were the mean ± standard deviation (SD) of 3 independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < .05; **P < .01). FITC, fluorescein isothiocyanate; Lyo, LyoVec (transfection reagent).