Fig. 5

Chromatin binding reduces IL-33 release during necrosis. a–c TE-7 pools with stable, Dox-inducible overexpression of wild-type (WT) or truncated (Trunc) IL-33 were treated for 4 h with 20 μM of calcium ionophore A23187 (Iono) or vehicle (Veh). a shows a representative western blot. b is quantification of the ratio of IL-33 detected in the supernatant vs. the pellet (S/P) by western blot. c is quantification of the percent release of lactate dehydrogenase (LDH) using enzymatic activity assay, defined as LDH activity in supernatant divided by the total amount in the supernatant plus pellet [S/(S+P)]. b, c depict the mean and standard error of the mean of three independent experiments. Two-way ANOVA interaction terms for IL-33 and % LDH release were p = 0.05 and p > 0.90, respectively. d–f The same pools of IL-33–overexpressing TE-7 cells were subjected to cryoshock. d shows a representative western blot. e, f are quantification of the ratio of IL-33 detected in supernatant to pellet (e) or cell viability as assessed by Trypan blue exclusion (f); presented is the mean and standard error of the mean of three independent experiments. ***, p < 0.001 and n.s.: not significant by Student’s t-test. ANOVA: analysis of variance, Dox: doxycycline, H3: histone H3, HSP90: heat shock protein 90, IL: interleukin, P: cell pellets, S: supernatants