(A) pJB908 plasmid extracted from cytosolic fractions of macrophages challenged with L. pneumophila (pJB908) WT or ⊿sdhA strains at 6 hpi.
(B) pJB908 plasmid extracted from cytosolic fractions of macrophages challengedwith WT L. pneumophila (pJB908) at3hpiinthe presence or absence of IFNβ 20 hr pre-treatment.
(C) Ifnb transcriptional induction from WT and L. pneumophila ⊿sdhA-infected macrophages of the indicated strains measured by qRT-PCR at 4 hpi.
(D) Cell death of B6, Casp11−/− and Casp1−/−Casp11−/− macrophages when challenged with L. pneumophila ⊿sdhA.
(E) Representative images of ⊿sdhA L. pneumophila and ASC immunofluorescence by antibody staining at 7 hpi. Images were taken with 20× lens; scale bar, 10 μm.
(F) Left: quantification of ASC-positive staining gated within regions of interest (ROIs), defined as regions of positive anti-L. pneumophila signal. Right: quantification of ASC-positive cellsfrom total numberofcells enumerated via Hoechst staining. Each symbol is a technical replicate. One representative experiment out of 3 is shown.
(G)Whole-cell lysate and precipitated supernatant from L. pneumophila-infected cells were probed by western blot for a p17 cleavage product of IL-1β Representative western blot on the left; quantification from n = 3 on the right.