Figure 5.

IL-1α release can occur independently of cell lysis. A–D, LPS-primed (1 μg/ml, 4 h) primary mouse BMDMs were incubated with vehicle, punicalagin (Pun; 50 μm), or glycine (Gly; 5 mm) 15 min prior to activation with ionomycin (10 μm, 1 h) or nigericin (10 μm, 1 h). A, cell death was measured as LDH release (n = 5). IL-1α release was assayed by ELISA (n = 5) (B) or analyzed for pro-IL-1α (31 kDa) and m-IL-1α (17 kDa) by Western blotting (C). D, IL-1β release was assayed by ELISA (n = 5). E, BMDMs were treated as previously but incubated with calpain inhibitor III (40 μm) or NBC6 (20 μm) prior to ionomycin or nigericin treatment. Combined supernatants and lysates were analyzed for GSDMD full-length (FL; 53 kDa), GSDMD N-terminal fragment (NT; 31 kDa), and β-actin (42 kDa) by Western blotting. *, p < 0.01; **, p < 0.001; ***, p < 0.0001; ****, p < 0.0001; n.s., nonsignificant, determined by one-way ANOVA with Dunnet's post hoc analysis compared with the ionomycin- or nigericin-treated groups. Western blots are representative of two independent experiments. Error bars, S.E.