Fig. 5. NLRP11 is necessary for oligomerization of human NLRP3.

a–c, Fluorescence microscopy of EGFP and DAPI in HEK293^NLRP3-EGFP cells transiently cotransfected with (a) ASC and NLRP11, (b) empty plasmid (Ctrl) and increasing concentrations of NLRP11 and (c) Ctrl, NLRP11, NLRP11^ΔPYD, NLRP11^PYD, NLRP11^ΔNACHT, NLRP11^NACHT, NLRP11^ΔLRR or NLRP11^LRR, as indicated (left) and presented as NLRP3 oligomer⁺ cells per view (right) (a, ASC: n = 4, ASC + NLRP11: n = 13; b, Ctrl n = 3, NLRP11^{lo, med}: n = 5, NLRP11^hi: n = 9; c: n = 5; mean ± s.d.; a, *P = 0.0003; b, *P = 0.0006, **P = 0.0164, ***P = 0.0009; c, *P < 0.0001; a–c, scale bars, 100 μm). d,e, Confocal microscopy of NLRP3 and DAPI staining, scale bars, 50 μm (d), and quantification of NLRP3 oligomer⁺ cells per view (e) using PMA-differentiated Cas9^Ctrl, NLRP3^KO, NLRP11^KO and NLRP11^KO restored with NLRP11-Flag or truncated NLRP11-Flag, as indicated primed with Pam3CSK4 (1 μg ml⁻¹, 4 h) and activated with nigericin (5 μM, 25 min) (n = 3 mean ± s.d.); *P < 0.0001; **P = 0.0001, ***P = 0.0002. f–h, Blue native PAGE and immunoblot for NLRP3 and Flag using TCLs from Cas9^Ctrl, NLRP3^KO, ASC^KD and NLRP11^Flag cells (f), Cas9^Ctrl, NLRP3^KO and NLRP11^KO cells (g) and NLRP11^Flag or NLRP11^ΔPYD-Flag cells (h) left untreated, primed with LPS (200 ng ml⁻¹, 4 h) (f,g) or Pam3CSK4 (1 μg ml⁻¹, 4 h) (h) and activated with nigericin (5 μM, 20–30 min). i, IL-1β ELISA using SNs from cells indicated above left untreated, primed with Pam3CSK4 (1 μg ml⁻¹, 4 h) and primed and activated with nigericin (5 μM, 25 min) (n = 3, mean ± s.d.); *P < 0.0001.

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