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Fig. 4
Intracellular LPS-triggered-caspy2 noncanonical inflammasome activation. a-c ZF4 cells were primed with Pam3CSK4 for 4 h, before being stimulated with cholera toxin B subunit (CTB) plus LPS, LPS, or CTB alone for 12 h. Relative caspase-5-like activity was measured by incubating cell lysates with fluorogenic and chromogenic caspase-5 substrates (WEHD) (a). Supernatants from the indicated HeLa cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release (b). Mixtures of cell lysates and supernatants were subjected to immunoblotting (c). d, e CASP4−/− HeLa cells were transduced with a vector expressing wild-type caspy2, caspy2 (C296A), caspy2 (ΔPYD), or the empty vector. Cells were primed with LPS for 4 h, before being stimulated with CTB plus LPS for 12 h. Images were taken as in Fig. 1d (d). Propidium iodide (PI) was added to detect the loss of plasma membrane integrity. Arrows denote cells with pyroptotic-like features. Scale bar, 50 µm. Relative caspase-5-like activity and LDH release assays were conducted as in a and b (e). Immunoblotting for the caspy2 forms indicated is shown. a–e Results are representative of at least three independent experiments, and error bars denote the SD of triplicate wells. *p < 0.05 (t test)