Fig. 2
The role of NLRP3 inflammasome in regulation of ZIKV-induced IL-1β secretion. a, b THP-1 macrophages were treated with Casp-1 inhibitor VX-765 for 1 h or 2 μM Nigericin for 2 h or infected with ZIKV at an MOI = 1 for 24 h. c–f THP-1 cells stably expressing shRNAs targeting NLRP3, ASC, or Casp-1 were generated and treated with 2 µM Nigericin for 2 h (c, d) or infected with ZIKV at an MOI = 1 for 24 h (e and f). IL-1β levels in the supernatants were determined by ELISA (a, c, e). p17 and p22/p20 levels in the supernatants were determined by western blot (b, d, f, top). NLRP3, ASC, pro-Casp-1, pro-IL-1β, Casp-1, and GAPDH proteins in the lysates were determined by western blot (b, d, f, bottom). (g–i) BMDCs prepared from the bone marrow (g, h) or BMDMs prepared from bone marrow cells (i) of treated C57BL/6 WT mice and C57BL/6 NLRP3−/− mice were stimulated with LPS (1 µg/ml) for 6 h and 5 mM ATP for 30 min or infected with ZIKV for 24 h at an MOI = 1. IL-1β levels in supernatants (g, i) and NLRP 3 proteins in lysates (h) were determined by western blot. (j–m) THP-1 macrophages were infected with ZIKV at an MOI = 1 for 24 h (j, m). HeLa cell line (k) and Vero cell line (l) that stably expressed NLRP3 were generated and infected with ZIKV at an MOI = 1 for 24 h. Subcellular localizations of NLRP3 (green) and ACS (green) and the nucleus marker DAPI (blue) were examined under confocal microscopy. Scale bar is 10 μm. n THP-1 macrophages were infected with ZIKV at an MOI = 1 for 24 h or treated with 2 μM Nigericin for 2 h. ASC oligomerization was determined by western blot using an anti-ASC antibody. The number of replicates is two (a, c, e, g, i). Data shown are means ± s.e.m; *P < 0.05, ***P < 0.0001 (one-way ANOVA with Tukey’s post-hoc test)
