1. Fig. 2. Spontaneous inflammasome activation by pyrin S242R.
Spontaneous inflammasome activation by pyrin S242R. (A) Confocal microscopy showing a pyrin-driven increase in cytoplasmic specks of GFP-tagged ASC when cotransfected into HEK293T cells with wild-type (WT) or mutated pyrin (S242R). Scale bar, 50 μm. (B) Representative fluorescence-activated cell sorting analysis of GFP-ASC specks with pyrin and pyrin S242R. (C) Quantification of three biological replicates for GFP-ASC specks by flow cytometry, with different pyrin mutants. (D) Western blots for active caspase-1 (p20 subunit) after expression of caspase-1 and ASC in HEK293T cells, together with an increasing concentration of Myc-tagged pyrin or pyrin (S242R). cDNA, complementary DNA. (E and F) Monocytic THP-1 cells where caspase-1 or pyrin was deleted by CRISPR, then reconstituted by retroviral expression of pyrin or pyrin (S242R). IL-1β was measured by enzyme-linked immunosorbent assay (ELISA) (E), and cell death was quantified by propidium iodide (PI) staining and flow cytometry 24 hours after stimulation with LPS (F). All are representative of at least three biologically independent experiments. Error bars represent means + SD for three biological replicates. ****P < Ctrl, control.
Seth L. Masters et al., Sci Transl Med 2016;8:332ra45
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