Fig. 2. GSDMD induces transient macrophage permeabilization upon infection.

a BMDMs from Gsdmd^–/– mice were transduced with a lentivirus encoding GSDMD-mNeon. Representative images of BMDMs expressing GSDMD-mNeon (green), pretreated for 4 h with LPS (100 ng/mL) and infected with L. amazonensis; MOI 3. Cell nucleus was marked with Dapi (blue), plasma membrane was marked with WGA (red). Yellow line indicates the scan analysis in the graph. Scale bar 20 µm. b BMDMs from WT, Nlrp3^–/–, Gsdmd^–/– and Casp1/11^–/– mice were pretreated for 4 h with LPS (100 ng/mL) and left non-infected (NI) or infected (MOI 10) for 4 h or 24 h. Cell permeabilization was assessed by flow cytometry upon propidium iodide (PI) staining. c–e BMDMs were pretreated for 4 h with LPS (100 ng/mL) and infected at an MOI 10 for 2 h or 24 h; the cultures were stained with PI (red) and Hoechst (blue) and assayed for pore formation by microscopy. d Representative images of pore formation. A total of 100 cells in each triplicate well were analyzed. Scale bar 20 µm. f Live-cell visualization of BMDMs from WT mice pretreated for 4 h with LPS (100 ng/mL) and with PI in the culture medium. Images (0, 10, and 20 h post infection/treatment) were acquired during a video (see Supplementary Movies 1–3). Scale bar 20 µm. g BMDMs were pretreated for 4 h with LPS (100 ng/mL) and left non-infected (NI), treated with nigericin (10 µM), or infected for 2 h and 24 h (MOI 10) to assess intracellular K⁺. Each bar represents the percentage of APG-2 fluorescence intensity in relation to the average fluorescence of non-infected (NI) cells. RFU, relative fluorescence units. Data are presented as mean values ± SD of triplicate wells. ^#P < 0.05 compared to NI; *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.