Extended Data Fig. 3: Bacterial mRNA and LPS are both required for IL-1β secretion and generation of the active form of caspase-11.
From: Caspase-11 interaction with NLRP3 potentiates the noncanonical activation of the NLRP3 inflammasome
a, Cytokine concentrations and LDH release in culture supernatants (20 h) post-transfection of WT or Nlrp3–/– macrophages as indicated with 2 ng ml–1 ultrapure LPS and/or 10, 30 and 100 ng ml–1 of mRNA prepared from E. coli LPSmut, L. innocua, or in vitro transcribed (IVT). b, Immunoblots of macrophage concentrated supernatants, WCE, mixed concentrated supernatants and WCE, or pulldown of caspases with biotinylated zVAD-FMK from WT, Nlrp3–/– or Casp11–/– macrophages 20 h post stimulation with L, HK or HK E. coli supplemented with E. coli total RNA (RNAtot), or transfection with indicated doses of ultrapure LPS and E. coli RNAtot. c, In vitro zVAD-AMC fluorescence post-incubation with immunoprecipitates of endogenous caspase-11 from macrophages stimulated for 6 h or 12 h as indicated with L, HK, or HK E. coli supplemented with E. coli RNAtot (10 µg ml–1). No IgG served as a control for Protein G-bound proteins alone. Bacteria:macrophage=20:1. Error bars, mean ± s.e.m. Results represent at least three independent experiments.
