Figure 4.

GSDME Is Processed in Caspase-1 Caspase-1/11-Independent Necrotic Cell Induced by NLRP3 Inflammasome Activation

(A) Primary peritoneal macrophages isolated from WT, Casp1/11^−/−, and Asc^–/–Casp1/11^−/− mice were rested or primed with Pam3CSK4 (100 ng/mL) for 18 h and then treated with nigericin (5μM) for 3 h. Lysates and supernatants were analyzed by western blot.

(B) Control, ASCKO, and CASP1 KO THP1 cells were differentiated with PMA for 48 h and treated with nigericin (5 μM) for 8 h. Lysates and supernatants were analyzed by western blot.

(C) Control, ASCKO, and CASP1KO THP1 NLRP3 D303N cells were differentiated with PMA for 48 h and treated with DOX (1 μg/mL) for 18 h. Lysates and supernatants were analyzed by western blot.

(D) Primed WT and Casp1/11^−/− macrophages were treated with nigericin for 3 h. Cells were lysed with Triton X-114 and separated into an aqueous phase and detergent phase. Each fraction was precipitated by acetone and analyzed by western blot.

(E and F) Pam3CSK4-primed WT macrophages were stimulated with nigericin. (E) After 3 h, cells were lysed with Triton X and supernatants were analyzed by western blot. (F) After 30 min, cell lysates were cross-linked with BS3 and analyzed by western blot.

(G) THP1 NLRP3 D303N/hIL1B cells were differentiated with PMA for 48 h and treated with DOX for 6 h. Lysates and supernatants were analyzed by western blot.

(A–G) Data are representative of two independent experiments.