Extended Data Fig. 1: PFO remodeled the TGN but not other organelles.
a, Coomassie blue staining for rPFO and rPFOY181A purified from E. coli. Arrows indicate rPFO and rPFOY181A proteins. Representative from at least 3 independent experiments. b, Representative LDH release assay of HeLa cells incubated with rPFO or rPFOY181A for 80 min (n = 3 wells per condition; mean ± s.d.; two-sided t-test). Representative from 3 independent experiments. c, Quantification of TGN dispersion (left) and NLRP3 recruitment (right) in HeLa cells stably expressing NLRP3-GFP incubated with rPFO or rPFOY181A for 80 min. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition; mean ± s.d.; two-sided t-test). NLRP3 recruitment was quantified from 100 cells (n = 3). Representative from 3 independent experiments. d, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) incubated with 10 μM nigericin, 0.18/0.54/0.90/1.8 nM rPFO or 0.18/0.54/0.90/1.8 nM rPFOY181A for 40 min. pro-Casp1 has two bands because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). SE, short exposure; LE, long exposure. Representative from 3 independent experiments. e, Representative fluorescence and phase contrast images in HeLa cells stably expressing TGN46-mScarlet-I incubated with 0.90 nM rPFO for 30 min. mScarlet-I was pseudocolored to green. The TGN46 vesicles in yellow frames were highlighted in the zoom-in images. Scale bar, 5 μm. Representative from 3 independent experiments. f, Representative immunofluorescence images (left) and quantification of GM130- or giantin-containing structure areas (right) in HeLa cells incubated with 0.90 nM rPFO or not (Mock) for 80 min. Scale bar, 10 μm. Areas containing the indicated organelle markers were measured with ImageJ (n = 40 cells per condition; NS, not significant). Representative from 3 independent experiments. g, Representative immunofluorescence images (left) and quantification of colocalization between rPFO-induced NLRP3 puncta and organelle markers (right) in HeLa cells stably expressing NLRP3-GFP incubated with 0.90 nM rPFO or not (Mock) for 80 min. Scale bar, 10 μm. Colocalization of NLRP3 puncta with the indicated organelle markers after rPFO treatment was analyzed with Pearson correlation coefficient using Coloc 2 plugin of ImageJ (n = 20 cells/sample; threshold regression: Costes). Representative from 3 independent experiments.
