Fig. 1: NLRP3 inflammasome assembly originates from PI4P-enriched endosomes.
From: Distinct changes in endosomal composition promote NLRP3 inflammasome activation
a, Confocal images of NLRP3-eGFP-expressing HeLa cells treated with vehicle (Ctrl), 15 µM nigericin or 45 μg ml−1 CL097 for 60 min. Cells were co-stained with antibodies against PI4P and EEA1. Magnifications of areas in dashed squares are shown in the lower left corner. Arrowheads indicate EEA1-positive endosomes containing PI4P and NLRP3-eGFP. Scale bar, 10 µm. b, Quantification of relative intensity of NLRP3-eGFP in PI4P+-, EEA1+- and PI4P+;EEA1+ structures in experiments shown in a. Mean ± s.d., N = 3, n = 20 cells for each group. c, Confocal images of PMA-differentiated ASC KO THP-1 cells expressing NLRP3-eGFP treated with vehicle (Ctrl), 15 µM nigericin or 50 µM CL097 for 30 min. Cells were co-stained with an antibody against EEA1. DAPI was used to stain the nucleus. Magnifications of areas in dashed squares are shown in the lower right corner. Arrowheads indicate EEA1-positive endosomes containing NLRP3-eGFP. Scale bar, 10 μm. d, Confocal images of PMA-differentiated ASC KO THP-1 cells expressing NLRP3-eGFP treated with vehicle (Ctrl), 15 µM nigericin or 50 µM CL097 for 30 min. Cells were co-stained with an antibody against PI4P. DAPI was used to stain the nucleus. Magnifications of areas in dashed squares are shown in the lower right corner. Arrowheads indicate PI4P-positive structures containing NLRP3-eGFP. Scale bar, 10 μm. e, Quantification of NLRP3-eGFP intensity on EEA1-positive endosomes in experiments shown in c. Mean ± s.d., N = 3, n = 20 cells for each group. f, Quantification of NLRP3-eGFP intensity on PI4P-positive vesicles in experiments shown in d. N = 3, n = 22 cells for each group. g, Z-stack maximal projection of confocal images of HeLa cells stably expressing ASCPYD-eGFP and NLRP3 treated with 10 µM nigericin for 20 min. Cells were stained with an antibody against EEA1. DAPI was used to stain the nucleus. Arrowheads show ASCPYD filaments originating from EEA1-positive endosomes. Scale bar, 0.5 μm. h, Quantification of ASCPYD filaments originating from EEA1-positive endosomes in experiments shown in g. Mean of percentage ± s.d., N = 3. Data were analyzed with an unpaired two-sided t-test (b, e, f, h). Data shown in a, c, d and g are representative of three independent experiments.
