Extended Data Fig. 9: DLY did not activate the NLRP3 inflammasome.
a, Representative LDH release assay of HeLa cells incubated with rDLY for 80 min (n = 3 wells per condition; mean ± s.d.). Representative from 2 independent experiments. b, Representative fluorescence and phase contrast images (left) and quantification of propidium iodide-positive cells (right) in HeLa cells incubated with rDLY or not (Mock) in 50 μg/mL propidium iodide-containing medium for 80 min. Scale bar, 25 μm. The percentage of cells with propidium iodide signal was quantified from 100 cells (n = 3; mean ± s.d.; two-sided t-test). Representative from 3 independent experiments. c, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) incubated with 10 μM nigericin or 0.18/0.54/1.8/5.4 nM rDLY 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). SE, short exposure; LE, long exposure. Representative from 3 independent experiments. d, Schematic for rPFO and rDLY chimeric proteins. The signal peptide (residue 1–28) of rPFO was deleted. rDLY does not have a signal peptide and therefore no deletion was performed. Domain (D)1, D2, D3, and D4 in rPFO are swapped with the corresponding domains in rDLY. D1, D2, and D3 are discontinuous domains. e, Representative LDH release assay of HeLa cells incubated with rPFO_DLYD4 (rPFO with its D4 replaced by D4 of rDLY) for 80 min (n = 3 wells per condition). Representative from 2 independent experiments. f, Representative immunofluorescence images (top) and quantification of TGN dispersion and NLRP3 recruitment (bottom) in HeLa cells stably expressing NLRP3-GFP incubated with 0.90 nM rPFO, 1.8 nM rPFO_DLYD4 or not (Mock) for 80 min. Scale bar, 10 μm. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition; NS, not significant). NLRP3 recruitment was quantified from 100 cells (n = 3; N.D., not detected). Representative from 3 independent experiments. g, Model: CDCs can be grouped into two types based on whether they can be internalized by host cells to remodel the TGN. Type A CDCs form pores on the plasma membrane before being internalized inside cells and translocating to the TGN. This translocation is mediated by D4 and is negatively regulated by Ca2+ influx-driven membrane repair, as membrane repair facilitates the clearance of CDC pores. After reaching the TGN, type A CDCs peel away the PtdIns4P-negative TGN membrane into multiple vesicles, thus exposing the remodeled perinuclear PtdIns4P-positive TGN membrane for NLRP3 inflammasome assembly. CDC-induced K+ efflux is not essential for TGN dispersion or NLRP3 recruitment but required for ASC recruitment. Type B CDCs (represented by DLY), while also capable of forming pores on the plasma membrane, do not get internalized. As a result, type B CDCs do not translocate to the TGN and therefore cannot remodel the TGN to activate the NLRP3 inflammasome.
