Mhc 1

BMDCs were extracted from the femurs and tibias of 7-week-old C57BL/6 mice and cultured for 8 days. Briefly, bone marrow cells were incubated in RPMI-1640 medium with 10% FBS and recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng/mL, PeproTech). On the 5th day, the medium was replaced with fresh medium with GM-CSF, and after 2 days the BMDCs were harvested for future use.
For the BMDC maturation assay, TC-1 cells (vector, NT-GSDMD) and BMDCs were cocultured in a 6-well plate at a ratio of 1:10 for 24 h, and doxy was added to induce pyroptotic cell death. The positive control BMDCs were cocultured with LPS (100 ng/mL). Next, the cocultured cells were harvested and stained with CD11c, CD86, CD80, MHC-I and MHC-II (Biolegend). Finally, flow cytometry was used to analyze the percentage of mature BMDCs.
For the BMDC migration assay using Transwells, GSDMD-NT-TC-1 cells were untreated or induced to undergo pyroptotic cell death with doxy for 24 h. Then, the supernatant was collected and placed in the bottom chambers of a 24-well Transwell plate and BMDCs were added to the upper chamber. The migration rate of the BMDCs was measured by optical microscopy after incubation for 6 h.

Total proteins were isolated from fresh macrophages cells (5.5 × 10⁶) or frozen exosomes (100 μg) were lysed using RIPA peptide lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins (30 μg) were loaded on 10% SDS-PAGE gels, electrophoresed, and then transferred to PVDF membranes (MilliporeSigma, Burlington, MA, United States). Subsequently, the membranes were incubated with primary antibodies against CD81 (at 1:2,000 dilution, ProteinTech Group, Inc., Chicago, United States), HSP70 (at 1:3,400 dilution, Biolegend, Inc., San Diego, CA, United States), LBP (at 1:2,400 dilution, ProteinTech Group, Inc.), CD36 (at 1:2,000 dilution, Biolegend, Inc.), MHC-I (at 1:3,000 dilution, Biolegend, Inc.) and TSG101 (at 1:2,000 dilution, ProteinTech Group, Inc.) at 4°C overnight. Then, PVDF membranes were washed three times with TBS-T under shaking. Finally, the membranes were incubated with secondary antibody solution (mouse anti-rabbit IgG-HRP at a 1:10,000) for 60 min at room temperature with gentle shaking, and washed with TBS-T under shaking three times again. Protein bands were imaged and analyzed using the Chemiluminescent Substrate System (Thermo Fisher Scientific, Inc., Waltham, MA, United States).

B16-OVA tumor cells infected with scramble sgRNA or sgRAD21 were stained with MHC-I (BioLegend, catalog 116525) and MHC-I SIINFEKL (eBioscience, catalog 17-5743-82). The B16-OVA or ID8-OVA tumor cells were cocultured with T cells (B3Z or OT-I cells) for 24 hours. The LacZ activity and supernatant levels of IL-2 and IFN-γ were examined as previously described (40 (link)). For intracellular cytokine staining, GolgiStop reagent (1,000×; BD Biosciences) was added to the coculture system for 3 hours before staining. First, OT-I cells were stained with fluorescence-labeled antibodies against CD8 (BioLegend, catalog 100706) for 1 hour at 4°C. Next, the cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer kit (eBioscience) and stained with anti–IFN-γ (eBioscience, catalog 17-7311-82) or anti-GZMB (eBioscience, catalog 12-8898-82). The stained cells were then analyzed using flow cytometry.