Extended Data Fig. 5: Endocytosis was not required for PFO trafficking or PFO-mediated NLRP3 inflammasome activation.
a–c, Representative immunofluorescence images (a), quantification of rPFO colocalization with the TGN (b) and TGN dispersion (c) in HeLa cells stably expressing scFv-sfGFP pre-treated with DMSO (solvent control), 25 µM retro-2cycl or pitstop 2 (20 µM) for 1 h before addition of 0.90 nM 10xGCN4-rPFO. Scale bar, 5 μm. Areas of rPFO foci colocalizing with the TGN were measured with ImageJ (n = 40 cells per condition; mean ± s.d.; two-sided t-test; N.D., not detectable, NS, not significant). Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition). Representative from 2 independent experiments. d, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) pre-treated with DMSO (solvent control) or 20 µM pitstop 2 for 1 h before addition of 0.90 nM rPFO for 40 min. pro-Casp1 has two bands in this cell line because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). Representative from 2 independent experiments.
