Fig. 2: HOIPINs inhibit PAMPs-induced activation of NF-κB and IFN antiviral pathways.
From: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses
a HOIPINs suppress the LPS-mediated NF-κB and IFN antiviral pathways. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 20 μg/ml LPS for the indicated period with HOIPINs. The cell lysates were immunoblotted with the indicated antibodies. b LPS-induced gene expression is suppressed by HOIPINs. BMDM cells were pre-treated with the indicated concentrations of HOIPINs for 30 min, and stimulated with 100 ng/ml LPS for 1 h. The mRNA levels were assessed by qPCR. c Suppression of IRF3 targets by HOIPIN-1. BMDM cells were stimulated with 100 ng/ml LPS for 8 h with HOIPIN-1, and interferon β (n = 13) and Cxcl10 (n = 10) were quantified by ELISA. d Suppression of antiviral signaling by HOIPIN-8. MEFs were stimulated with 10 μg/ml poly(I:C) for the indicated period with HOIPIN-8, and subjected to immunoblotting analysis. e Suppression of IRF3 targets by HOIPINs. BMDM cells were pre-treated with 30 μM HOIPINs for 30 min, and stimulated with 10 μg/ml poly(I:C) for 2 h with HOIPINs. The mRNA levels were assessed by qPCR. f HOIPIN-1 inhibits ISRE-luciferase activity. MEF cells, transfected with the ISRE-luciferase reporter, were stimulated with 10 μg/ml poly(I:C) with or without HOIPIN-1 for 6 h, and the luciferase activities were analyzed. g LUBAC activity is indispensable for the IFN pathway. WT- and HOIP−/−-MEFs were treated with 10 μg/ml poly(I:C) and HOIPIN-1 for 2 h. Cell lysates were subjected to immunoblotting analysis. h HOIP is critical for the expression of IRF3-target genes. WT- and HOIP−/−-MEFs were treated as in g, and qPCR analyses were performed. i The Sendai virus (SeV)-induced antiviral response is suppressed by HOIPINs. MEFs were infected with SeV at a multiplicity of infection (MOI) of 10 for 8 h, and treated with the indicated concentrations of HOIPINs for 30 min. qPCR analyses were performed. In b, c, e, f, h, i, data are shown as mean ± SEM, n = 3 (sample numbers in c are indicated in the legend). NS not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s post hoc test.
