Fig. 3: GSDMD is acetylated at Lysine 248.

HEK293T cells were transfected with GSDMD-p30 mutants (A) or GSDMD-FL mutants and Caspase-1 (B) or GSDMD-FL mutants and Caspase-4 (C). The release of LDH in the culture medium was detected. D Acetylation of GSDMD mutants expressed in HEK293T cells. PMA-differentiated GSDMD-KO THP-1 macrophages were infected with Flag-GSDMD-FL-WT/K248R-Lentivirus, followed with LPS and nigericin treatment. The supernatants were collected and analyzed by LDH release assay (E) and ELISA for IL-1β (F). G Domain organization and ribbon diagrams of GSDMD-WT (left) and GSDMD-K248Ac-K (right). The acetylated GSDMD structure was constructed using the Structure Editing module of UCSF Chimera and optimized using Minimize Structure. H Immunofluorescence microscopy and nuclear staining (with the DNA-binding dye DAPI) of HEK293T cells transfected with expression plasmids for Myc-GSDMD-WT or Myc-GSDMD-K248R. Scale bars, 20 μm. **** stands for P < 0.0001, *** stands for P < 0.001, ** stands for P < 0.01, ^* stands for P < 0.05 (unpaired t test). Data shown are mean ± SD from one representative experiments performed in triplicate.