a, b, Preparation of the GSDMA3(N + C) used for conjugation onto the nanoparticle. Purified GSDMA3 containing a engineered PPase cleavage site between the gasdermin N and C domain was cleaved in vitro to obtain GSDMA3(N + C). a, Coomassie blue staining of the prepared GSDMA3 proteins. b, ATP-based viability of CT26 cells electroporated with the prepared GSDMA3 protein. Mean ± s.d., n = 3 independent replicates. c, d, Pore-forming activity of the gasdermin N domain released from NP–GSDMA3 by desilylation catalysed by Phe-BF 3 . c, Representative negative-stain electron microscopy images of the gasdermin pores formed by purified native GSDMA3(N + C) protein or the GSDMA3(N + C) protein released from NP–GSDMA3. Scale bars, 100 nm. d, Representative 2D averages of the gasdermin pores viewed from the top (left) and the side (right). Scale bar, 10 nm. e, f, HeLa or EMT6 cells were treated with NP–GSDMA3 alone or in combination with Phe-BF 3 . e, Total cell lysates or their membrane fractions were immunoblotted with indicated antibodies. Flag–GSDMA3(N + C) proteins (the Flag tag was fused C-terminal to the gasdermin N domain, before the PPase cleavage site) were conjugated to the nanoparticle. The GSDMA3 N domain was probed by anti-Flag immunoblotting. f, LDH assay of the effect of various inhibitors on cell death induced by treatment with NP–GSDMA3 and Phe-BF 3 . Mean ± s.d., n = 3 independent replicates. Fer1, ferrostatin 1; Nec-1, necrostatin 1. g, HeLa, EMT6 or 4T1 cells, or primary BMDMs, were treated as indicated. NP + GSDMA3, nanoparticles mixed with the noncovalent GSDMA3(N + C) complex. GSDMA3(Mut), the pore forming-deficient E14K/L184D mutant version of GSDMA3(N + C). Flow-cytometry plots of PI- and annexin V–-FITC-stained cells are shown. h, Effect of phagocytosis inhibitor cytochalasin D on cell death induced by treatment with NP–GSDMA3 and Phe-BF 3 in primary BMDMs. Flow-cytometry plots of PI- and annexin V–Alexa-647-stained cells are shown. Data are representative of two (c–f, h) or three (a, b, g) independent experiments. Source Data