Fig 2: Schematic illustration of self-adaptive pyroptosis-responsive nanoliposomes (RC-NL@DMF) blocking pyroptosis in autoimmune inflammatory diseases. A) Structure of RC-NL@DMF and characterization of its pyroptosis-responsive drug-release functionality; B) RC-NL@DMF treats pyroptosis-related autoimmune inflammatory diseases by suppressing pyroptosis in macrophages and attenuating inflammation resulting from pyroptosis; and C) Schematic illustration of pyroptosis-responsive pore formation for drug release in the pyroptotic cells. This objective is spirited into three folds: 1) Pyroptosis-responsive liposomes efficiently penetrate the cells, facilitated by the action of the R8 cell-penetrating peptide; 2) Once pyroptosis occurs, the activated GSDME-N selectively binds to the CL on the liposome's surface, creating pores for the encapsulated drug release; and 3) DMF, a classical pyroptosis inhibitor, exerts its pyroptosis-inhibiting effects and subsequent inflammatory response attenuation by inhibiting the caspase 3/GSDME pathway.

Fig 3: Effect of apoptosis inhibitors/enhancers on muNS processing. (A) Effect of Q-VD-OPh on muNS processing. 35S-radiolabeled in-vitro-synthesized muNS is incubated for 4 h at 37 °C with extracts from ARV-infected cells that have been incubated (lane 2) or not (lane 1) with 10 μM Q-VD-OPh from the onset of the infection. These samples, as well as an extract of ARV-infected CEF immunoprecipitated against muNS (lane 3), are analyzed by 10% SDS-PAGE and autoradiography. (B) Effect of apoptotic enhancers and inhibitors on muNS processing and apoptosis in DF1 cells. DF-1 cell monolayers are infected with 10 PFU/cell of ARV S1133. The cells in lanes 3 and 5 are treated at the onset of the infection with 10 μM Q-VD-OPh. The cells in lanes 2 and 3 are treated from 10 to 16 hpi with 1 μg/ml actinomycin D, and in lanes 4 and 5 with 0.5 μM staurosporine. Caspase 3/7 activity, DNA damage and muNS processing are determined at 16 hpi as for Fig. 2A–C. (C) Effect of apoptotic enhancers and inhibitors on muNS processing in transfected cells. CEF monolayers are transfected with the pCINeo-muNS plasmid and 24 h later the cells are incubated for 6 h with the same compounds as for Fig. 3B. The cells are then lysed and the resulting extracts subjected to Western blot analysis with anti-muNS serum. The positions of nonstructural viral proteins are indicated on the left and that of protein markers on the right.

Fig 4: Conservation and functional importance of S/N234 in CASP3/7.(A) Weblogo analysis of the conservation of S/N234 in CASP3/7 in Mammalia, Aves, Reptilia, Amphibia, and Osteichthyes. (B) Sequence alignment of HsCASP3/7. The S/N residues are indicated by red arrow. (C) HsGSDME was treated with HsCASP3 or HsCASP3-N208S for 2 hr, and then subjected to immunoblotting with anti-HsGSDME-CT antibody. (D) HsGSDME was co-expressed in HEK293T cells with different doses of HsCASP3 or HsCASP3-N208S for 24 hr. The cell lysates were immunoblotted to detect HsGSDME, CASP3, and β-actin. (E) Sequence alignment of the S/N234 region in the CASP7 of primate (shaded cyan) and non-primate mammals. The S/N residues are indicated by red arrow. Asterisks indicate identical residues. (F) Alignment of human and mouse CASP7 sequences. The S/N234 residues are indicated by arrow. (G) HsGSDME was treated with different units of HsCASP7 or MmCASP7 for 2 hr, and the cleavage was assessed by immunoblotting with anti-HsGSDME-CT antibody. (H) HsGSDME was incubated with different units of MmCASP7 or its mutant (N234S) for 2 hr, and HsGSDME cleavage was analyzed as above. (I) MmCASP7 was treated with HsCASP7, MmCASP7, or MmCASP7-N234S for 2 hr. The cleavage was determined by immunoblotting with anti-MmGSDME-NT antibody. For panels (C, D, G–I), FL, full length; NT, N-terminal fragment; CT, C-terminal fragment. Figure 6—source data 1.Original file for the western blot analysis in Figure 6. Figure 6—source data 2.File containing Figure 6C, D–G, and I and original scans of the relevant western blot analysis with highlighted bands.

Fig 5: TGF-β2-induced apoptosis of ORS cells, keratin exposure was generated from TGFβ2-induced apoptotic ORS cells, and keratin release from TGFβ2-mediated apoptotic ORS cells induced DP cell condensation.a Graphical quantification of apoptosis array of ORS cells and TGFβ2-treated ORS cells. b TGFβ2-induced apoptosis and its following keratin exposure of ORS cells by immunofluorescent staining; DAPI, blue; phalloidin, keratin 34, red; annexin V, keratin 34, TUNEL, caspase 3, green. Scale bars, 200 μm. c Images of DP cell condensation on TGFβ2-treated ORS cell layers. Co-culture of cell tracker-treated DP cells (red) on TGFβ2-treated ORS cell layers. Immunofluorescent image; E-cadherin, green; DAPI, blue. Scale bars, 200 μm. d DP cell condensation under conditioned medium collected from TGFβ2-treated ORS cell culture. Western blot image of released keratin 34 from ORS cell culture and TGFβ2-treated ORS cell culture. Immunofluorescent image; ALPase, red; β-catenin, green; DAPI, blue; Control-DP medium, DP culture medium; Control-ORS Medium-TGFβ2, ORS medium including TGFβ2; CM from TGFβ2-treated ORS, conditioned medium collected from TGFβ2-treated ORS cell culture; Keratin treatment, DP medium containing 1(w/v)% keratin. Scale bars, 50 μm.