Fig. 3: GSDMD is important for inflammasome activation in Leishmania-infected macrophages.

a, b Bone marrow-derived macrophages (BMDMs) generated from C57BL/6 (WT) and Gsdmd^–/– mice were pretreated for 4 h with LPS (100 ng/mL) and infected with L. amazonensis at an MOI 10 for 2 h or 24 h. The cultures were stained with anti-ASC (green, indicated by arrows), and cell nuclei were stained with DAPI (blue). a Representative images of ASC puncta formation in response to L. amazonensis infection. Images were acquired by fluorescence microscopy with a ×100 oil immersion objective and analyzed using ImageJ software. Scale bar 20 µm. b The percentage of ASC puncta in BMDMs infected by L. amazonensis for 2 or 24 hs. Nigericin was used as a positive control. A total of 100 cells in each triplicate well were analyzed. c Lysates from C57BL/6 (WT) and Gsdmd^−/− macrophages were pretreated for 4 h with LPS (100 ng/mL) and infected with L. amazonensis for 24 h assessed for ASC oligomerization by western blot. ASC monomers, dimers, trimers, and oligomers are indicated in the figure. ns non-specific band. d BMDMs from WT, Gsdmd^–/–, Nlrp3^–/–, and Casp1/11^–/– mice were treated with LPS (100 ng/mL) for 4 h and infected with L. amazonensis at an MOI of 10 for 24 h. IL-1β production was measured by ELISA. e Western blot analyses of lysate and supernatant of WT and Gsdmd^–/– BMDMs pretreated for 4 h with LPS (100 ng/mL) and were left non-infected (NI) or infected by Leishmania at an MOI 10 for 2 h or 24 h. The unprocessed (31 kDa) and processed (17 kDa) IL-1β are indicated in the figure. MW molecular weight. Data are presented as mean values ± SD of triplicate wells. ^#P < 0.05 compared with NI cells; *P < 0.05 comparing the indicated groups, as determined by two-way ANOVA. Shown is one representative experiment of five independent experiments performed. Source data are provided as a Source Data file.