Extended Data Fig. 5: Palmitoylation promotes plasma membrane translocation of GSDMD-NT.

a, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GSDMD-NT were incubated with the indicated concentrations of Dox in the presence or absence of 2-BP (50 μM) for 12 hours. Cell lysates were then collected and subjected to Western blot analyses with anti-GSDMD antibodies. b, c, HEK293T cells were transfected with GSDMD-NT (b) or GSDMD and caspase-1 (c) in the presence or absence of 2-BP (50 μM) for 24 hours. The distribution of GSDMD-NT in subcellular fractions was determined by Western blot analyses with anti-GSDMD antibodies. d, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT were incubated with or without Dox (2 μg/ml) for indicated times. Cell death was measured by SytoxGreen positivity assay. Data are presented as mean ± s.d. of n = 4 independent wells of one representative experiment. e, Flow cytometry gating strategy. Representative schema describes the gating to identify specific GFP⁺ BMDMs. Dox-inducible GFP–GSDMD-NT-reconstituted BMDMs were stimulated with DOX (2 µg/ml) for 12 hours to produce GFP fluorescence. Single cell suspensions were first gated using forward scatter/side scatter (FSC vs SSC) to exclude debris (Gate 1), then gated using Green-B to identify GFP⁺ cells (Gate 2). f, g, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT (WT or C192A) were incubated with Dox (2 μg/ml) in the presence or absence of 2-BP (50 μM) for 12 hours, then flow cytometry analysis of GFP activity was performed. h, i, Gsdmd-deficient BMDMs reconstituted with Dox-inducible GFP–GSDMD-NT (WT or C192A) were incubated with or without Dox (2 μg/ml) in the presence or absence of 2-BP (50 μM) for 12 hours. Fluorescent images of GFP–GSDMD-NT (green) translocation from the cytoplasm to the basal plasma membrane were determined by TIRF microscopy. Images shown are representative of three independent experiments.

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