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. 2016 Aug 30;26(9):1007–1020. doi: 10.1038/cr.2016.100

Figure 2.

Figure 2

GSDMD-N translocates to plasma membrane in pyroptotic cells. (A) Deconvolution microscopy of MLKL-HA-expressing RAW-asc cells treated with DMSO or TSZ for 5 h. After treatment, cells were immunostained for HA and counterstained with Hoechst and PI. PI positive indicates dead cell. (B) Plasma membrane marker Hras-RFP was expressed and detected in MLKL-HA-expressing RAW-asc cells. Cells were treated and analyzed as in A. (C) Deconvolution microscopy of HA-GSDMD-reconstituted Gsdmd−/− RAW-asc cells primed with LPS for 4 h and subsequently treated with or without Nig for 2 h. (D) The same as in C except that plasma membrane marker Hras-RFP was expressed and detected in HA-GSDMD-reconstituted RAW-asc cells. (E) Gsdmd−/− RAW-asc cells were reconstituted with GSDMD-Flag, then treated and analyzed as in C. (F) Quantification of cells in C with uniformly diffused GSDMD (UD), plasma membrane enriched GSDMD plus PI-positive (PM, PI+) and plasma membrane enriched GSDMD plus PI-negative (PM, PI−). (G) Purified recombinant GST-GSDMD-N and GST-GSDMD-C were incubated with a general lipid strip, respectively. Protein-lipid binding assay was performed as described in the Material and Methods. (H) Raw-asc cells were primed with LPS for 4 h and then pretreated with 3MA (10 mM), YM201636 (80 μM), SF1670 (2 μM) or PITenin-7 (20 μM) for 1 h. Cells were then treated with Nig for 2 h and cell survival was determined by PI staining. Results shown are mean ± SD representative of three independent experiments. Scale bar, 5 μm.