Fig. 3. GSDME cleavage during programed spontaneous neutrophil death was mediated by PR3 and caspase-3.

a GSDME cleavage in neutrophils treated with caspase inhibitors. Neutrophils from WT mouse bone marrow were treated with pan-caspase (QVD-OPH 100 μM), caspase-3 (Z-DEVD-FMK 100 μM), caspase-1 (VX765 25 μM), caspase-8 (Z-IETD-FMK 10 μM), or caspase-9 (Z-LEHD-FMK 20 μM) inhibitors for 16 h. β-actin was used as a protein loading control. Results are representative of three biological replicates. Relative levels of cleaved GSDME-NT were quantified based on densitometry. Results are means (±SD). n = 4 independent repeats. **P < 0.01 versus DMSO-treated neutrophils by two-sided Student’s t-test (QVD-OPH, P = 0.0001; Z-DEVD-FMK, P = 0.0029). b GSDME cleavage in neutrophils treated with serine protease inhibitor diisopropyl fluorophosphate (DFP). Neutrophils were treated with serine protease DFP (100 μM) or Z-DEVD-FMK (as a positive control) for 6 h. Representative immunoblots of three independent experiments are shown. The densitometry results are the means (±SD) of three independent experiments. *P < 0.05 versus DMSO-treated neutrophils by two-sided Student’s t-test (DFP, P = 0.022; Z-DEVD-FMK, P = 0.0211). c Cleavage of GSDME by serine proteases and caspase-3. Mouse GSDME was overexpressed in HEK293T cells. Cell lysates were incubated with the indicated proteases or caspase-3 for 1 h. GSDME cleavage was assessed by immunoblotting. UT untreated, EL elastase, CG cathepsin G, PR3 proteinase 3. Results are representative of three independent experiments. d Summary of GSDME cleavage during programed spontaneous neutrophil death. In aging neutrophils, PR3 is released from the granules, leading to cleavage and activation of procaspase-3. Active caspase-3 in turn cleaves GSDME to generate GSDME-NT, which targets the plasma membrane to induce membrane pore formation and lytic cell death. e GSDME cleavage in neutrophils during inflammation. Mice were challenged with heat-inactivated E. coli. (HIEC, 10⁷ cfu) for indicated time periods. GSDME cleavage in purified neutrophils was assessed by western blotting as described above. Representative immunoblots of three independent experiments are shown. The densitometry results are the means (±SD) of three independent experiments. Source data are provided as a Source Data file.