Fig. 3

GSDME physically interacts with EGFR. A Diagram of the GSDME structure and the caspase-3 cleavage sites (Asp²⁷⁰). B A549 cells were treated with 0, 1, 2, 4 or 8 μM doxorubicin for 24 h, and GSDME-FL and GSDME-N were detected by Western blot analysis. GSDME-FL, full-length GSDME; GSDME-N, the N-terminal cleavage products of GSDME. C Interaction network of GSDME (also named DFNA5) analyzed in the BioGRID database. D HEK293FT cells were transfected with pcDNA3.1, pcDNA3.1-FLAG-EGFR, pcDNA3.1-FLAG-GSDME (D270A)-HA or pcDNA3.1-FLAG-GSDME-HA plasmids for 24 h. A co-IP assay was performed with HA antibody, and the coeluted proteins were detected by Western blot analysis with EGFR and HA antibodies. E HEK293FT cells were transfected with pcDNA3.1, pcDNA3.1-HA-EGFR, pcDNA3.1-FLAG-GSDME-N or pcDNA3.1-FLAG-GSDME-HA plasmids for 24 h. A Co-IP assay was performed with a FLAG antibody, and the coeluted proteins were detected by Western blot analysis with HA and FLAG antibodies. F H1299 cells were transfected with pcDNA3.1, pcDNA3.1-FLAG-GSDME-HA or pcDNA3.1-FLAG-GSDME (D270A)-HA plasmids for 24 h and treated with or without 4 μM doxorubicin for 8 h. A Co-IP assay was performed with HA antibody, and the coeluted proteins were detected by Western blot analysis with EGFR and GSDME antibodies. G Schematic diagram of EGFR. ETTM, extracellular and transmembrane fragment; CTTM, cytoplasmic and transmembrane fragment; CT, cytoplasmic fragment. H The interaction of GSDME with different fragments (CTTM, CT, ETTM) of EGFR was detected by a co-IP assay in HEK293FT cells. A co-IP assay was performed with HA antibody, and the coeluted proteins were detected by Western blot analysis with FLAG and HA antibodies. β-Actin was used as the loading control for the input. I Molecular docking analysis for GSDME and the cytoplasmic domain of EGFR using the ClusPro 2.0 server