MMAE specifically increased cGAMP-mediated immune response in a STING-dependent manner.

Abstract

(A) THP1-Lucia ISG cells (WT and STING KO) were treated with cGAMP and/or MMAE for 6 h, respectively. Total RNA was harvested and IFNβ, CXCL10, CCL5, TNFα, IL-6, ISG15, IFIT3 and IFITM1 mRNA expression was measured by real-time PCR (n = 3 biological replicates). (B and C) THP1-Lucia ISG cells (WT and MyD88 KO) were stimulated with cGAMP with or without MMAE for 24 h (B) or 6 h (C), and luciferase signals were measured (B). Phosphorylation of STING downstream signal transduction was assessed by immunoblotting with indicated antibodies (C). (D-G) THP1-Lucia ISG cells were stimulated with cGAMP, Sendai virus (SeV, MOI = 0.1), LPS (1 μg), Poly(I:C) (10 μg) or co-treated with indicated concentrations of MMAE for 24 h. ISRE reporter activity was measured and the fold changes in luminescent signals were normalized to DMSO-treated cells. (H and I) THP1-Lucia NF-kB cells were treated with LPS (1 μg), IL-1β (1 μg) or combined with MMAE for 24 h. Fold changes in NF-kB activation were measured by Lucia luciferase signal and normalized to DMSO-treated cells. Bars are the mean ± SEM of indicated (n) independent experiments. Significance was determined by one-way ANOVA; *p p p p (TIF)</p