Figure 5. Polyubiquitinated IL-1β from colonic IECs is released via a caspase-8–dependent nonlytic pathway.

(A and B) Gsdmd^–/– YAMC cells were stably restored with either WT or D276A GSDMD (Gsdmd^D276A), and stimulated as indicated. (A) Supernatants from stimulated cells were subjected to IP with anti–IL-1β (WCLs were directly analyzed). (B) Total protein in cell supernatants was extracted and analyzed, alongside WCLs. (C) Pore-forming ability was assessed by kinetic analysis of propidium iodide (PI) uptake in LPS- and ATP-stimulated WT and Gsdmd^–/– YAMC cells, with triton treatment used as a positive control. Error bars are shown as SEM of technical replicates (n = 3). (D) IL-1β–deficient (IL-1β–KO) and WT control YAMC cells were left untreated or stimulated with LPS followed by ATP, and analyzed for indicated genes. (E) WT YAMC cells transduced with scramble (Scr) or caspase-8 targeting (Casp8 KD) shRNA were subjected to co-IP with anti–IL-1β. Precipitated supernatant proteins and WCLs were analyzed by Western blot. (F) WT YAMC cells were subjected to co-IP with anti-GSDMD and Western analysis as indicated. All experiments were repeated 3 times and yielded consistent results. All error bars show SEM with *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed unpaired Student’s t test.