Journal: Nature immunology

Article Title: Interleukin-6 signaling promotes alternative macrophage activation to limit obesity-associated insulin resistance and endotoxemia

doi: 10.1038/ni.2865

Figure Lengend Snippet: (a) Heatmap of differentially regulated transcripts between IL-6-stimulated (4 hours) bone marrow-derived macrophages (BMDM) either from control (Ctrl) or Il6ra Δmyel ( Il6ra −/− ) mice. (n=4 per group; fold-change cut off ± 1.25; p≤0.05). (b) qRT-PCR analyses of Il6ra and Il4ra expression in untreated (NT) and IL-6-stimulated (12 hour) Ctrl and Il6ra −/− BMDM (n=6 per group; *p≤0.001 vs Ctrl; **p≤0.001 vs NT; Data are expressed as % of NT Ctrl). (c) FACS analysis of untreated (NT) and IL-6-stimulated (24 hour) Ctrl BMDM (n=5; *p≤0.001 vs NT). (d) Ctrl BMDM were left untreated (NT) or stimulated with IL-6 (12 hours) in the absence (IgG) or presence of an IL-10-neutralizing antibody (αIL-10) and qRT-PCR analysis of Il4ra expression was performed (Data is representative of two independent experiments; Data are expressed as % of NT Ctrl). (e) Immunoblot of Ctrl BMDM that were left untreated (NT) or stimulated with IL-6 (12 hours) in the presence of a control (IgG) or IL-10-neutralizing antibody (αIL-10) (n=3). (f) qRT-PCR analyses of Stat3 and Il6ra expression in siRNA-transfected Ctrl BMDM (n=3; *p≤0.001; Data are expressed as % of Ctrl siRNA). (g) qRT-PCR analyses of Il4ra expression in untreated (NT) or IL-6 stimulated (4h) siRNA-transfected Ctrl BMDM (n=3; *p≤0.001 vs IL-6 stimulated Ctrl siRNA; Data are expressed as % of NT Ctrl siRNA). (Values are expressed as mean ± sem)

Article Snippet: Bone marrow-derived macrophages were stimulated with recombinant IL-6 (R&D), IL-4, IL-10 (both from PeproTech) or E. coli -derived lipopolysaccharides (LPS O55:B5; Sigma).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Western Blot, Transfection

Journal: Nature immunology

Article Title: Interleukin-6 signaling promotes alternative macrophage activation to limit obesity-associated insulin resistance and endotoxemia

doi: 10.1038/ni.2865

Figure Lengend Snippet: (a) Luciferase activity of the indicated reporter constructs in untreated (NT) or IL-6-treated (12h) immortalized macrophages. (n=3; *p≤0.001 vs NT −1,300 bp fragment). (b) qRT-PCR analyses of ChIP assay showing occupancy of p-STAT3 over the Il4ra promoter in control (Ctrl) and Il6ra −/− BMDM stimulated with IL-6 for the indicated time points (n=3 vs 3; *p≤0.001 vs Ctrl; **p≤0.05, ***p≤0.001 vs NT; Data are expressed as % of NT Ctrl). (Values are expressed as mean ± sem)

Article Snippet: Bone marrow-derived macrophages were stimulated with recombinant IL-6 (R&D), IL-4, IL-10 (both from PeproTech) or E. coli -derived lipopolysaccharides (LPS O55:B5; Sigma).

Techniques: Luciferase, Activity Assay, Construct, Quantitative RT-PCR

Journal: Nature immunology

Article Title: Interleukin-6 signaling promotes alternative macrophage activation to limit obesity-associated insulin resistance and endotoxemia

doi: 10.1038/ni.2865

Figure Lengend Snippet: (a) Immunoblot of control (Ctrl) and Il6ra −/− BMDM that were left untreated or pre-incubated with IL-6 (12 hours) before stimulation with IL-4 (30 minutes) (Representative immunoblot of 3 independent experiments shown). (b ) qRT-PCR analyses of Ctrl and Il6ra −/− BMDM that were stimulated with IL-6 (12 hours) and subsequently exposed to IL-4 or IL-4 + IL-6 for 24 hours (n=6 vs 6; *p≤0.01 vs Ctrl; **p≤0.001 vs NT; Data are expressed as % of NT Ctrl). (c) FACS analysis of CD206/MRC1 and ARG1 expression in Ctrl BMDM that were left untreated or stimulated with IL-6, IL-4 or IL-4 + IL-6 (n=5; *p≤0.05 vs IL-6 + IL-4; **p≤0.001). (d) FACS analysis of CD206/MRC1 expression in WAT and BAT (n=5; *p≤0.05 vs IL-6+IL-4; **p≤0.01 ***p≤0.001) or (e) blood and peritoneum of Ctrl mice that were treated with IL-6, IL-4 or IL-4 + IL-6 (n=5; *p≤0.05 vs IL-6+IL-4; **p≤0.01). (f) GTT area under the curve (AUC) of HFD Ctrl and Il6ra Δmyel mice before (NT) and after a 4-week treatment period with IL-4 (n=15 vs 17; *p≤0.05 vs Ctrl; **p≤0.01). (g) qRT-PCR analyses of HFD Ctrl and Il6ra Δmyel mice treated with IL-4 for 4 weeks (n=7–8 vs 7; *p≤0.05; **p≤0.01 ***p≤0.001; Data are expressed as % of NT Ctrl). (h) Immunohistochemical staining of CD206/MRC1 in livers from HFD Ctrl and Il6ra Δmyel mice that were left untreated (NT) or treated with IL-4 for 1 week (Representative images of 3 per group shown; scale bars represent 50μm).

Article Snippet: Bone marrow-derived macrophages were stimulated with recombinant IL-6 (R&D), IL-4, IL-10 (both from PeproTech) or E. coli -derived lipopolysaccharides (LPS O55:B5; Sigma).

Techniques: Western Blot, Incubation, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining

Journal: Nature immunology

Article Title: Interleukin-6 signaling promotes alternative macrophage activation to limit obesity-associated insulin resistance and endotoxemia

doi: 10.1038/ni.2865

Figure Lengend Snippet: (a) qRT-PCR analyses of control (Ctrl) and Il6ra −/− BMDM that were left untreated or stimulated with IL-6 (12 hours) and subsequently exposed to bacterial lipopolysaccharides (LPS) alone or LPS + IL-6 for an additional 24 hours (n=6 vs 6; *p≤0.05 vs LPS + IL-6 stimulated Ctrl; **p≤0.05, ***p≤0.01; Data are expressed as % of LPS stimulated Ctrl). (b) Body weight loss of Ctrl and Il6ra Δmyel mice 24 hours after exposure to 1mg/kg LPS (n=7 vs 8; *p≤0.05). (c) Food intake of Ctrl and Il6ra Δmyel mice before (basal) and 24 hours after exposure to LPS (n=7 vs 8; *p≤0.01). (d) Respiratory exchange ratio (RER) of Ctrl and Il6ra Δmyel mice before (basal) and 24 hours after exposure 1mg/kg LPS (n=7 vs 8; *p≤0.01). (e) Cytokine concentration in circulation of Ctrl and Il6ra Δmyel mice 6 hours after exposure to 1mg/kg LPS (n=7 vs 7; **p≤0.01, p=0.06 for IL-4). (f) qRT-PCR analyses of liver and WAT from Ctrl and Il6ra Δmyel mice 48 hours after exposure to 1mg/kg LPS (n=7 vs 8; *p≤0.05; Data are expressed as % of Ctrl). (Values are expressed as mean ± sem).

Article Snippet: Bone marrow-derived macrophages were stimulated with recombinant IL-6 (R&D), IL-4, IL-10 (both from PeproTech) or E. coli -derived lipopolysaccharides (LPS O55:B5; Sigma).

Techniques: Quantitative RT-PCR, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Structural basis for the interaction of protein S1 with the Escherichia coli ribosome

doi: 10.1093/nar/gku1314

Figure Lengend Snippet: Free S1 NTS binds to the ribosome and interferes with translation of the canonical ompA mRNA. ( A ) Purified S1-depleted 30S ribosomes (30S(-S1)) were incubated in the absence (lanes 1 and 2) or in the presence of FITC labelled S1 NTS (lanes 7 and 8), native protein S1 (lanes 9 and 10) or both (lanes 11 and 12). Likewise, FITC labelled S1 NTS (lanes 3 and 4) or native S1 (lanes 5 and 6) were incubated in the absence of ribosomes. Before (input; lanes 1, 3, 5, 7, 9 and 11) and after ultrafiltration using 100 kDa MWCO Amicon concentrators (Millipore) samples were taken and the presence of the respective proteins and the S1 NTS peptide in the ribosome fraction (ribosome fraction; lanes 2, 4, 6, 8, 10 and 12) was determined by SDS-PAGE. ( B ) In vitro translation of ompA mRNA in the absence (lane 1) or in the presence of a 10- or 50-fold molar excess over ribosomes of S1 NTD (lanes 2 and 3), S1 D1 (lanes 4 and 5) or S1 NTS (lanes 6 and 7), respectively. The assay was performed in triplicate and one representative autoradiograph is shown. Graph representing the quantification of three independent assays is given below. Error bars represent the standard deviation of the mean.

Article Snippet: The purified proteins were concentrated using Amicon filter devices (MWCO of 3 kDa; Millipore).

Techniques: Purification, Incubation, SDS Page, In Vitro, Autoradiography, Standard Deviation