Gene induction profiles of CVB3 CL and VSV hairpin.

<p>A549 cells were transfected with indicated concentrations of CVB3 CL or VSV hairpin for 24-PCR analysis using the BioMark system. Data presented as fold of induction in log10 scale as compared to mock-treated sample.</p

ivtRNA of the first 1-I.

<p>ivtRNAs with sequences corresponding to the indicated positions of CVB3 genomic RNA were transfected into MEFs from MDA5^−/− (B), RIG-I^−/− (D), IPS-I^−/− (F)mice or their WT litter controls (A, C, E). IFN-β luciferase reporter assay was carried out 8 hr.p.t.. Data presented as mean +/− standard deviation (SD). (G) ivtRNAs with sequences corresponding to the indicated positions of CVB3 genomic RNA were transfected into WT or RIG-I^−/− MEFs. IFN-β luciferase reporter assay was carried out 8 hr.p.t.. Data presented as mean +/− SD.</p

CVB3 CL activates IFN and ISG as efficiently as published ligand.

<p>(A) CVB3 CL (with and without CIP treatment), VSV hairpin and a short dsRNA control of 100 bp) were analyzed on an 8 M Urea/8% Acrylamide gel, and visualized with SYBR Gold staining. (B) A549 cells were transfected with indicated amounts of VSV short hairpin RNA or CVB3 CL RNA. Cells were lysed at 24 hr.p.t. and subjected SDS-PAGE analysis followed by immunoblotting using antibodies against the indicated proteins. (C, D) A549 cells were transfected with 0.1, 1 or 10 ng/ml of VSV short hairpin RNA or CVB3 CL RNA and incubated for 24 hrs. Total RNA was isolated and subjected to qRT-PCR analysis using primers specific for IFNβ or ISG56 mRNAs. Data presented as mean +/− SD.</p

In vitro transcribed RNAs of the first 2-I.

<p>(A) Schematic representation of known RNA structures in CVB3 genomic RNA. CL, cloverleaf. IRES, internal ribosomal entry site. CRE, <i>cis</i>-acting RNA element. Numbers refer to nucleotide positions in the viral genomic RNA. (B, C) In vitro transcribed (ivt) RNAs with sequences corresponding to the indicated positions of CVB3 genomic RNA were transfected into WT (B) or RIG-I^−/− (C) MEFs. IFN-β luciferase reporter assay was carried out 8 hours post transfection (hr.p.t.). Data presented as mean +/− SD.</p

CVB3 CL protects cells against DENV and VSV infections.

<p>(A) A549 cells transfected with 0.1, 1, 10 or 100 ng/ml RNA constructs for 18 hrs prior to DENV challenge at MOI of 0.5. Cells were fixed 24 hrs after infection and stained with an antibody against the viral envelop (E) protein. DENV E+ cells were quantified and presented as percentage of the total population. Data plotted as mean +/− SD. (B) A549 cells were transfected with 0.1, 1, 10 or 100 ng/ml RNA constructs for 18 hrs prior to VSV-GFP challenge at MOI of 1. GFP-positive cells were quantified at 24 hrs post infection and presented as percentage of the total population. Data plotted as mean +/− SD. (C) HeLa cells were transfected with 1, 10 or 100 ng cloverleaf (CL) or Domain III (DIII) and incubated for 16 hr. They were then infected with enterovirus (EV) 71 at an MOI of 0.05 for 8 hrs. Virus titers were determined by end point titration on HeLa cells. Data shown as mean +/− SD.</p

CVB3 Cloverleaf is a potent RIG-I agonist and protects against subsequent EV71 infection.

<p>(A, B) ivtRNAs with sequences corresponding to the indicated positions of CVB3 genomic RNA were transfected into WT MEFs. IFN-β luciferase reporter assay was carried out 8 hr.p.t.. Data presented as mean +/− SD. (C) ivtRNAs of individual domains in CVB3 5′ UTR were transfected into WT MEFs. IFN-β luciferase reporter assay was carried out 8 hr.p.t.. Data presented as mean +/− SD. (D) Schematic representation of RNA ligands used in C. (E) CVB3 CL was mock-treated or treated with calf intestinal phosphatase (CIP), and transfected into RIG-I^+/− or RIG-I^−/− MEFs. IFN-β mRNA levels were determined at 8 hr.p.t.. Data presented as mean +/− SD. (F) RNAs used in E were analyzed on an 8 M Urea/8% Acrylamide gel, and visualized with SYBR Gold staining. RNA fragments were transfected at equimolar amounts in each experiment.</p

MAP3K8 in humans is associated with higher BMI and cytokine expression.

<p>MAP3K8 mRNA expression in human subcutaneous adipose tissue, associated with (a) BMI, (b) plasma insulin values, (c) plasma glucose levels, (d) HOMA-IR, (e) adipocyte sell size cell size and (f) crown-like structures. *p<0.05. n = 51, 50, 71, 70 respectively. HOMA-IR  =  Homeostatic Model Assessment for insulin resistance.</p

Obesity and macrophage influx in adipose tissue of HFD-fed WT and MAP3K8-ko animals.

<p>MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a) Bodyweight development upon LFD or HFD feeding. (b) Epididymal white adipose tissue (eWAT) weight after 16 weeks of LFD or HFD. (c) Liver weight after 16 weeks of LFD or HFD. (d) Plasma CXCL1 levels after 16 weeks of LFD or HFD (e) Macrophage influx into the adipose tissue as determined by immunohistochemistry, F4/80 (serotec) staining: 20× magnification or 40× as indicated: (f) Number of crown-like structures per field. (g–i) qPCR analysis for macrophage infiltration markers, (g) CD68, (h) F4/80, (i) MCP-1 in adipose tissue of MAP3K8-ko and WT animals. * p<0.05, ** p<0.01, *** p<0.001.</p

Inflammatory profile of the adipose tissue of HFD-fed WT and MAP3K8-ko animals.

<p>MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a–f) qPCR analysis for cytokines (a) TNF-α, (b) IFNγ, (c) IL-1β, (d) CXCL-1, (e) IL-6 and (f) IL-1Ra. n = 9 mice per group. Relative phosphorylation of NFκB p65 (g) and ERK 1/2 (h) in eWAT of MAP3K8-ko and WT animals after HFD-feeding (i). * p<0.05, ** p<0.01, *** p<0.001.</p

MAP3K8-ko mice display similar bodyweight and insulin sensitivity compared to WT mice.

<p>MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a) Plasma insulin and (b) plasma glucose levels after diet intervention. Insulin (itt) and oral glucose (ogtt) tolerance tests after 16 weeks of diet intervention. (c) itt after 16 weeks of HFD and (d) area under the curve itt. (e) ogtt after 16 weeks of HFD and (f) area under the curve of ogtt. n = 9 mice per group. * p<0.05, ** p<0.01, *** p<0.001.</p