Methods
time-lapse fluorescence microscopy (video imaging), colorimetry, and ELISA. Cleaved forms of GSDMD, and caspase-1 were detected by Western blotting. Treatment of THP-1 cells with rmCIRP increased MET formation as revealed by SYTOX Orange Staining assay in a time- and dose-dependent manner. METs formed by rmCIRP stimulation were further confirmed by extracellular DNA, citrullinated histone H3, and myeloperoxidase. Treatment of THP-1 cells with rmCIRP significantly increased the cleaved forms of caspase-1 and GSDMD compared to PBS-treated cells. Treatment of macrophages with caspase-1, and GSDMD inhibitors z-VAD-fmk, and disulfiram, separately, significantly decreased rmCIRP-induced MET formation. We also confirmed rmCIRP-induced MET formation using primary cells murine peritoneal macrophages. These data clearly show that eCIRP serves as a novel inducer of MET formation through the activation of GSDMD and caspase-1.