Fig. 2. NLRP11 is required for NLRP3-mediated caspase-1 activation.

a–d, Flow cytometry of fluorochrome-labeled inhibitors of caspases assay (FLICA) signals plotted versus side scatter area (SSC-A) in Ctrl^KD and NLRP11^KD cells (a,b) and primary human macrophages transfected with Ctrl^siRNA or NLRP11^siRNA (c) or Ctrl^Myc and NLRP11^Myc cells left untreated, primed with LPS (1 μg ml⁻¹, 1 h) and primed and activated with nigericin (5 μM, 45 min) (a,c), nigericin (5 μM, 15 min) (d) or transfected with poly(dA:dT) (6 ng ml⁻¹, 4 h) (b). e, Immunoblot for cleaved and total caspase-1, cleaved and total GSDMD and tubulin loading control from SN and TCL of Cas9^Ctrl, NLRP11^KO and CASP1^KO cells left untreated, primed with Pam3CSK4 (1 μg ml⁻¹, 4 h) and primed and activated with nigericin (5 μM, 30 min). The arrowhead marks the cleaved GSDMD fragment also detected by the total GSDMD antibody. f, Immunoblot for cleaved and total caspase-1 using SNs and TCLs of Cas9^Ctrl and NLRP11^KO cells left untreated, primed with Pam3CSK4 (1 μg ml⁻¹, 2 h) and primed + transfected with poly(dA:dT) (2 μg ml⁻¹, 6 h). g, LDH release from Cas9^Ctrl, NLRP11^KO and NLRP3^KO cells primed with LPS (200 ng ml⁻¹, 4 h) or primed and activated with nigericin (5 μM, 30 min) is presented as percent cytotoxicity compared to maximum LDH release (n = 3, mean ± s.d.); *P = 0.0002, **P = 0.0001. h, Immunoblot for ASC and NLRP3 using SN and TCL of Ctrl^KD and NLRP11^KD cells primed with LPS (200 ng ml⁻¹, 4 h) or primed and activated with nigericin (5 μM, 15 min).

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