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. Author manuscript; available in PMC: 2022 Oct 29.
Published in final edited form as: Nat Immunol. 2022 Apr 29;23(5):705–717. doi: 10.1038/s41590-022-01192-4

Figure 4. Requirement of NLRP3 and ASC for caspase-11 activation in response to live avirulent Gram-negative bacteria.

Figure 4.

a-c, Immunoblots of macrophage concentrated supernatants (20hr) or WCE (6hr), and cytokine concentrations and LDH release in culture supernatants (20hr) post-stimulation of WT, Nlrp3–/– or Pycard–/– macrophages with L or HK E. coli (a), WT, Nlrp3–/– or Pycard–/– macrophages with L or HK avirulent or virulent E. coli (b), and WT, Nlrp3–/– or Ipaf–/– macrophages with Live Salmonella ΔSpi1/2 (avirulent, lacking the Salmonella pathogenicity islands 1 and 2 encoded type III secretion system) or WT (virulent) or Shigella BS103 (avirulent virulence plasmid cured) or WT (virulent) (note the doublet GSDMD bands here likely reflect the nature of the trigger) (c). d, Immunoblot of WT macrophage cross-linked fractions (16hr) post-stimulation with L, HK or HK E. coli supplemented with indicated doses of RNAtot, Flagellin or poly(dA:dT). e, Immunoblots of WT macrophage concentrated supernatants (20hr) or WCE (6hr), and cytokine concentrations in culture supernatants (20hr) post-stimulation as in d. LDH measured by cytotoxicity assay; IL-1β and IL-6 by ELISA. Error bars, mean ± s.e.m. One-way ANOVA followed by multiple comparisons Sidak tests and p values indicated in bar graphs in a, b, c and e (n=3). ns: non-significant. Bacteria:macrophage=20:1 for E. coli and virulent E. coli, 5:1 for all Salmonella and Shigella strains. Results represent at least 3 independent experiments.