Fig. 4. NLRP3 activators result in retention of TGN46 on endosomes.

a, Confocal images of HeLa cells stably expressing eGFP-tagged NLRP3 and mApple-tagged RAB5A. Cells were treated with vehicle (Ctrl), 10 μM nigericin for 40 min or 45 μg ml⁻¹ CL097 for 80 min and stained with an antibody against TGN46. DAPI was used to stain the nucleus. Magnifications of areas in dashed squares are shown in the lower right corner. Arrowheads indicate RAB5-positive endosomes containing TGN46 and NLRP3-eGFP. Scale bar, 10 μm. b, Quantification of the number of RAB5-positive endosomes containing NLRP3-eGFP and TGN46 in experiments shown in a. Mean ± s.d., N = 3, n = 34 cells for vehicle (Ctrl) group; n = 47 cells for nigericin-treated group and n = 44 for CL097-treated group. c, Confocal images of HeLa cells expressing NLRP3-eGFP before and after treatment with 15 µM nigericin for the indicated times. Cells were stained with an antibody against TGN46. Scale bar, 10 µm. d, Confocal images of HeLa cells expressing HA-tagged TGN46 (TGN46-HA) before and after treatment with 15 µM nigericin for the indicated times. Cells were stained with antibodies against PI4P and HA tag. Scale bar, 10 µm. e, Upper panels: Quantification of temporally resolved nigericin-treated HeLa cells showing peripheral PI4P (left), NLRP3 puncta formation (middle) and TGN46 dispersion (right) in experiments shown in c and d. Mean of percentage ± s.d., N = 4, n = 100 cells for each group. Lower panels: Quantification of numbers of peripheral PI4P spots (left), NLRP3 puncta (middle) and TGN46 peripheral distribution (right) in each cell during nigericin treatment in experiments shown in c and d. Mean ± s.d., N = 3, n = 20 cells for each group. Data were analyzed with an unpaired two-sided t-test (b, e). Data shown in a, c and d are representative of at least three independent experiments.