Fig. 5

OGT functioned as a suppressing factor for GSDME cleavage via increasing its O-GlcNAcylation. (A) Western blot was applied to identify the impacts of OGT overexpression and knockdown on the OGT and total O-GlcNAc levels, as well as O-GlcNAcylation modifications of NLRP3, caspase 1, GSDMD and GSDME. (B) The O-GlcNAcylation sites of GSDME predicted by GlycoMine website. (C, D) To ascertain the site of O-GlcNAcylation modification on GSDME, potential sites were mutated and then OGT, total O-GlcNAc, GSDME O-GlcNAcylation were assessed by western blot. (E) The phosphorylated and cleaved GSDME levels mediated by OGT were also determined with western blot. RL-2, O-GlcNAc; si-nc, small interfering RNA negative control; si-OGT, small interfering RNA targeting OGT; WT, wild-type; p-GSDME(Thr6), phosphorylation of GSDME at Thr6 site; GSDME-N, GSDME-N-terminal domain. ^**P < 0.01. ^*P < 0.05. ns: no significance