Involvement of MAPK activation in IL-1β-induced IL-36γ expression.

<p>(A) MAPK activation in colonic SEMFs. The cells were stimulated with IL-1β (10 ng/ml), and the activation of MAPKs were analyzed by Western blotting. Antibodies against phosphorylated (p)- and total- MAPKs were used. *P < 0.05, **P<0.01. (B) The cells were stimulated for 24 h with IL-1β (10ng/ml) in the presence or absence of MEK inhibitors [U0216 (10 μM) and PD98059 (10 μM)] and a p38 inhibitor [SB203580 (10 μM)]. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments).</p

Effects of IL-1β on IL-36γ mRNA expression in colonic myofibroblasts.

<p>(A) Dose-dependent effects of IL-1β on IL-36γ mRNA expression. The cells were incubated for 24 h with increasing concentrations of IL-1β. IL-36γ mRNA expression was expressed relative to β-actin mRNA expression (mean ± SD from 4 different experiments). (B) Time-dependent effects of IL-1β on IL-36γ mRNA expression. The cells were stimulated with IL-1β (10 ng/ml) for the pre-determined times. IL-36γ mRNA expression was expressed relative to β-actin mRNA expression (mean ± SD from 4 different experiments). (C) Dose-dependent effects of IL-1β on IL-36γ secretion. The cells were incubated for 24 h with increasing concentrations of IL-1β. IL-36γ level in supernatant was determined by ELISA (mean ± SD from 4 different experiments). (D) Time-dependent effects of IL-1β on IL-36γ secretion. The cells were stimulated with IL-1β (10 ng/ml) for the pre-determined times. IL-36γ level was determined by ELISA (mean ± SD from 4 different experiments). *P<0.05, **P<0.01 versus medium; ANOVA followed by Bonferroni’s post hoc test.</p

Combined effects of IL-1β and other cytokines.

<p>(A) The cells were incubated for 24 h with combination of IL-1β (10 ng/ml) and other cytokines [TNF-α (100 ng/ml), IFN-γ (100 ng/ml), IL-4 (100 ng/ml), and/or IL-17A (100 ng/ml)]. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments). **P<0.01 versus IL-1β alone. (B) The cells were stimulated for 24 h with or without IL-1β (10 ng/ml), TNF-α (100 ng/ml), and/or combination of IL-1β (10 ng/ml) and TNF-α (100 ng/ml), and intracellular IL-36γ was analyzed by Western blot.</p

Involvement of activation of transcription factors, NF-κB and /AP-1, in IL-1β-induced IL-36γ expression.

<p>(A) IL-1β induced IκBα phosphorylation and degradation in colonic myofibroblasts. The cells were stimulated with IL-1β (10 ng/ml), and phosphorylated IκBα were detected by Western blotting. (B) IL-1β induced activation of NF-κB and c-Jun AP-1. The cells were stimulated with IL-1β (10 ng/ml), and NF-κB p65 and phosphorylated c-Jun were detected by immunocytochemistory. Reacted antibodies against NF-κB p65 were visualized by FITC (green fluorescence)-labeled second antibody. Reacted antibodies against phosphorylated c-Jun were detected by a DyLight^® 594 (red fluorescence)-labeled secondary antibodies. Nucleus was stained by DAPI (blue). (C) The cells were stimulated with IL-1β (10 ng/ml) or medium alone for 15 min and nuclear proteins were extracted. NF-κB p65 and phosphorylated c-Jun in nuclear extracts were detected by immunoblot. (D) Effects of silencing of NF-κB p65 and c-Jun AP-1on IL-1β-induced IL-36γ expression. The cells were transfected with control siRNA, the siRNA specific for NF-κB p65 and/or c-Jun AP-1, and incubated for 24h. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments). *P<0.05, **P < 0.01 versus IL-1β stimulation.</p