Journal:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining

Journal: Nature metabolism

Article Title: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

doi: 10.1038/s42255-019-0044-9

Figure Lengend Snippet: Fig. 2 | High-fat feeding increases Igfbp7 expression in LMs. a, Diagram showing genes with significant differences in expression in mice with HFD versus ND feeding; n = 3 mice per condition. P value was calculated by Wald test with DESeq2. To set the significance threshold, the adjusted P value was calculated by the Benjamini–Hochberg method with FDR < 0.05. b, Comparison of Igfbp7 expression (normalized RPKM) in mouse liver and LMs; n = 3 mice per condition. c, IGFBP7 protein expression in mouse LMs; n = 3 pooled mice/condition. d, IGFBP7 secretion from mouse LMs assessed by ELISA; n = 5 mice per condition. e, Igfbp7 expression in different cell types in mice; the number of mice (n) is indicated. Hep, hepatocytes; ATMs, adipose tissue macrophages; PM, peripheral macrophages. f, RNA ISH combined with immunohistochemistry (IHC) for mouse liver (scale bars, 20 μm); n = 3 independent experiments with similar results. EC, endothelial cell; H, hepatocyte. g, IGFBP7 RNA editing in human LMs. AA, amino acid. h, Representation of the positions at which RNA editing occurs with respect to the IGFBP7 protein domains. Data are shown as the mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t test.

Article Snippet: Cells were then subjected to a 1-h preincubation with U0126, an inhibitor of the upstream kinase activating ERK1/2, MAPK kinase (MEK) (10 mM concentration; Cell Signaling Technology, 9903), or maintained in the same medium and subsequently treated with recombinant IGFBP7 (20 ng/ml; R&D Systems, 2120-B7-025) for 4 h. Cell pellets were washed and RNA was extracted.

Techniques: Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Two Tailed Test

Journal: Nature metabolism

Article Title: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

doi: 10.1038/s42255-019-0044-9

Figure Lengend Snippet: Fig. 4 | Silencing of Igfbp7 in LMs decreases hyperglycaemia and hepatic steatosis. a–d, Glucose tolerance tests (a), overnight fasting glycaemia (b), insulin tolerance tests (c) and liver TG levels (d) in mice treated with GeRP-Scr or GeRP-Igfbp7; the number of mice (n) is indicated. e, Representative images of Oil Red O staining performed in mice treated with GeRP-Scr (n = 4) or GeRP-Igfbp7 (n = 5) (scale bars, 100 μm); five random images were taken per mouse with similar results. f,g, Liver transmission electron microscopy (TEM) images (f) and lipid droplet size quantification (g). h, LM and hepatocyte TEM (blue arrowheads indicate lipid droplets); for all TEM experiments, 15 images were randomly acquired from 3 different mice treated with GeRP-Scr or GeRP-Igfbp7. i, NPC LipidTOX staining measured by flow cytometry; n = 5 mice per condition. MFI, median fluorescence intensity. j, Gene set enrichment analysis depicting up- and downregulated pathways in hepatocytes from mice treated with GeRP-Scr (n = 3) or GeRP-Igfbp7 (n = 4) according to MES. The x axis indicates the rank of the pathways according to MES. P value was calculated by permutation test. To set the significance threshold, adjusted P value was calculated by the Benjamini–Hochberg method with FDR < 0.1. k,l, Gene expression pattern of selected genes (normalized RPKM) (k) and gene expression by qPCR (l) in isolated hepatocytes from mice treated with GeRP-Scr or GeRP-Igfbp7; the number of mice (n) is indicated. DNL, de novo lipogenesis; HGP, hepatic glucose production. m, Images of ImpL2-Gal4>UAS-mCherry flies showing ImpL2+ cells (red), pHrodo Green staining (green) and overlay (merge). Images to the right represent flies fed a ND or HFD. The green channel was used to visualize structure and for orientation; n = 8 independent experiments with similar results. n,o, ImpL2+ haemocytes counted in 15 randomly selected flies per condition (n) and ImpL2 expression in n = 6 flies per condition (o). p,q, Glucose (p) and TG (q) levels in n = 9 flies per condition. r, Immune gene expression in haemocytes; n = 6 flies per condition. s,t, Glucose (s) and TG (t) levels in ImpL2-RNAi (Hml-Gal4>ImpL2-RNAi) versus W (Hml-Gal4 × w) Drosophila fed a HFD; the number of flies (n) is indicated. A.u., arbitrary units. Data are shown as the mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t test in all panels except o, where one-way ANOVA was used with adjustment for multiple comparisons. DNL, de novo lipogenesis; LD, lipid droplets; HGP, hepatic glucose production.

Article Snippet: Cells were then subjected to a 1-h preincubation with U0126, an inhibitor of the upstream kinase activating ERK1/2, MAPK kinase (MEK) (10 mM concentration; Cell Signaling Technology, 9903), or maintained in the same medium and subsequently treated with recombinant IGFBP7 (20 ng/ml; R&D Systems, 2120-B7-025) for 4 h. Cell pellets were washed and RNA was extracted.

Techniques: Staining, Transmission Assay, Electron Microscopy, Flow Cytometry, Fluorescence, Gene Expression, Isolation, Expressing, Two Tailed Test

Journal: Nature metabolism

Article Title: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

doi: 10.1038/s42255-019-0044-9

Figure Lengend Snippet: Fig. 5 | LM-derived IGFBP7 increases lipogenesis and gluconeogenesis. a, Western blots of AKT phosphorylation in primary mouse hepatocytes treated with recombinant IGFBP7 (rIGFBP7) and short-term insulin (Ins); n = 4 biological replicates. p473AKT, AKT phosphorylated at residue 473. b, Blood glucose levels for fasted and refed states in mice fed a ND and treated with GeRP-Scr or GeRP-Igfbp7; the number of mice (n) is indicated. c, Western blots of immunoprecipitations in primary mouse hepatocytes for IR, IRS1 and IRS2; n = 3 biological replicates. p-Tyr, phosphorylated tyrosine. d, Western blots and quantification (for a and d) of AKT phosphorylation in primary mouse hepatocytes treated with IR antagonist (IRa) s961, recombinant IGFBP7 and short-term insulin; n = 4 biological replicates. e, Western blots and quantification of AKT phosphorylation in primary mouse hepatocytes treated with recombinant IGFBP7 under hyperinsulinaemia; n = 3 biological replicates. f, Western blots and quantification showing immunoprecipitation of IGFBP7 with IR under hyperinsulinaemia; n = 3 biological replicates. g,h, Western blots and quantification of phosphorylated ERK1/2 (pERK1/2) (g) and immunoprecipitation of Shc with IR (h) in primary mouse hepatocytes treated with recombinant IGFBP7 under hyperinsulinaemia; n = 3 biological replicates. i, Gene expression in primary mouse hepatocytes treated with insulin, recombinant IGFBP7 and the ERK pathway inhibitor U0126 (ERKi); n = 10 technical replicates. j, Image of a human liver spheroid (scale bar, 200 μm). k, Western blots and quantification of AKT and ERK1/2 phosphorylation in human liver spheroids treated with short-term (7-min) or long-term (4-h) insulin and with wild-type (wt) or edited (ed) recombinant IGFBP7 for 4 h. Quantification is expressed as fold change in comparison to control. l, Western blots and quantification of coimmunoprecipitation of IGFBP7 with IR in human liver spheroids under hyperinsulinaemia. Quantification is expressed as fold change in comparison to control. Data are shown as the mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t test in i and by one-way ANOVA with adjustment for multiple comparisons in b and d–h.

Article Snippet: Cells were then subjected to a 1-h preincubation with U0126, an inhibitor of the upstream kinase activating ERK1/2, MAPK kinase (MEK) (10 mM concentration; Cell Signaling Technology, 9903), or maintained in the same medium and subsequently treated with recombinant IGFBP7 (20 ng/ml; R&D Systems, 2120-B7-025) for 4 h. Cell pellets were washed and RNA was extracted.

Techniques: Derivative Assay, Western Blot, Phospho-proteomics, Recombinant, Residue, Immunoprecipitation, Gene Expression, Comparison, Control, Two Tailed Test