Figure 3. NLRP3 inflammasome assembly requires bacterial mRNA with LPS, irrespective of LPS stimulatory activity.

a-c, Immunoblots of macrophage cross-linked fractions and matching WCE (16hr), post-stimulation with L, HK or HK E. coli and E. coli^LPSmut + mRNA from each bacterium (a), L, HK or HK Y. pestis grown at 24°C or 37°C + Y. pestis RNA_tot (b), L, HK or HK E. coli + E. coli RNA_tot (c). d,e, Immunoblots of macrophage concentrated supernatants (20hr) or WCE (6hr), and cytokine concentrations and LDH release in culture supernatants (20hr) post-stimulation with L, HK or HK E. coli or E. coli^LPSmut +100ng/mL ultrapure LPS or 100ng/mL mRNA from each bacterium (d), and L, HK or HK Y. pestis grown at 24°C or 37°C +Y. pestis RNA_tot (e). LDH measured by cytotoxicity assay; IL-1β, TNF and IL-6 by ELISA. f,g, Immunoblots of macrophage cross-linked fractions and matching WCE as in a, post-stimulation with L, HK or HK E. coli+IVT or L. innocua mRNA (f), and L, HK or HK E. coli+E. coli or L. innocua RNA_tot, or E. coli mRNA or eukaryotic mRNA (g). h,i, Immunofluorescence confocal microscopy on macrophages stimulated for 16hr with L or HK Y. pestis grown at 24°C or 37°C (h), and L, HK or HK E. coli^LPSmut + E. coli^LPSmut RNA_tot or untreated (i). Bar graphs in h,i, % cells exhibiting ASC speck. Inserts, magnification of indicated areas. White arrowheads, ASC specks. Phalloidin delineates the macrophage actin cytoskeleton. Scale bar=10μm. j,k, Immunoblots of macrophage cross-linked fractions and matching WCE as in a, post-stimulation with L, HK or HK E. coli or L. innocua + E. coli or L. innocua RNA_tot with LPS or Lipid IVa as indicated. Error bars, mean ± s.e.m. One-way ANOVA followed by multiple comparisons Sidak tests and p values indicated in bar graphs in d (IL-1β: n=3, LDH: n=4), e (n=4), h (n=10) and i (Unst.: n=6, L: n=8, HK and HK+RNA: n=11). ns: non-significant. All macrophages are WT except as indicated in (c). Bacteria:macrophage=20:1. Results represent at least 3 independent experiments.