Figure 3.

NLRP3 inflammasome activation by DENV-2 viroporin, NS2A, and NS2B. HMEC-1 cells were transfected with the expression plasmid encoding GFP-tagged DENV-2 NS2A, NS2B, or the pEGFPN1 empty vector for 36 h or treated with LPS (1 μg/mL) for 6 h followed by ATP (5 mM) for 45 min as a positive control. Cell lysates were analyzed by western blot using (A) an anti-NLRP3 antibody (1:500), (B) an anti-ASC antibody (1:1,000), and (C) an anti-caspase-1 antibody (1:1,000). (D) HMEC-1 cells were transfected with the expression plasmid encoding GFP-tagged DENV-2 NS2A, NS2B, or the pEGFPN1 empty vector for 36 h or treated with LPS (1 μg/mL) for 6 h followed by ATP (5 mM) for 45 min as a positive control, and the cell free supernatant was analyzed for IL-1β by ELISA. (E) HMEC-1 cells were transfected with expression plasmids encoding GFP-tagged DENV-2 NS2A, NS2B, or the pEGFPN1 parental vector for 36 h or treated with LPS (1 μg/mL) for 6 h followed by ATP (5 mM) for 45 min as a positive control, and the cells were stained with anti-ASC (Red) and analyzed by a confocal microscope. (F) The number (#) of ASC puncta per cell was counted by confocal microscopy. ASC puncta was calculated from a total of 20 cells. (G) HMEC-1 cells were transfected with pNS2A-GFP, or the pEGFPN1 empty vector for 36 h and the cells were stained with anti-NLRP3 (1:200) (Red) and analyzed by a confocal microscope. Nuclei were visualized by staining with DAPI. (H) Pearson's Coefficient of the NS2A-GFP or GFP co-localization with NLRP3. Data are representative of at least three independent experiments, and indicate the mean ± S.D. (D,F,H). ^**P < 0.01 and ^***P < 0.001.