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: Recombinant human IGFBP7 (R&D Systems, 1334-B7-025) at a concentration of 100, 50, 25, 12.5, 3.1 and 1.5 ng/ml in PBS was also added as a standard.

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: Recombinant human IGFBP7 (R&D Systems, 1334-B7-025) at a concentration of 100, 50, 25, 12.5, 3.1 and 1.5 ng/ml in PBS was also added as a standard.

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: Recombinant human IGFBP7 (R&D Systems, 1334-B7-025) at a concentration of 100, 50, 25, 12.5, 3.1 and 1.5 ng/ml in PBS was also added as a standard.

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

Journal: Clinical Science (London, England : 1979)

Article Title: IGFBP7 promotes endothelial cell repair in the recovery phase of acute lung injury

doi: 10.1042/CS20240179

Figure Lengend Snippet:

Article Snippet: Recombinant Human IGFBP-7 protein , Elabscience , Cat# PDMH100099.

Techniques: Ubiquitin Proteomics, Recombinant, SYBR Green Assay, CCK-8 Assay, Extraction, Proliferation Assay, Immunoprecipitation

Journal: British Journal of Cancer

Article Title: Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

doi: 10.1038/sj.bjc.6605937

Figure Lengend Snippet: Protein sequence and surface plasmon resonance analysis of the anti-IGFBP7 sdAb 4.43. ( A ) Protein sequence of anti-IGFBP7 sdAb 4.43; CDR1, CDR2 and CDR3 are underlined. ( B ) Sensogram overlay showing 4.43 monomer binding to immobilised IGFBP7 at concentrations of 25, 25, 50, 50, 75, 75, 100 and 200 n M . ( C ) Fitting of equilibrium binding data to a steady-state affinity model. ( D ) Scatchard plots of the equilibrium binding data.

Article Snippet: Briefly, ∼1400 RU of recombinant human IGFBP7 (R&D Systems, Minneapolis, MN, USA) and 900 RU of ovalbumin (Sigma) as a reference protein were immobilised on a CM5 sensor chip using the amine-coupling kit supplied by the manufacturer.

Techniques: Sequencing, SPR Assay, Binding Assay

Journal: British Journal of Cancer

Article Title: Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

doi: 10.1038/sj.bjc.6605937

Figure Lengend Snippet: Representative immunofluorescence images demonstrating IGFBP7 immunoreactivity detected with the anti-IGFBP7 sdAb 4.43 in tissue sections of the ( A ) mouse orthotopic GBM, ( B ) contralateral healthy mouse brain and ( C ) human GBM. IGFBP7 immunoreactivity is shown in red (middle panels); the staining for the endothelium-specific markers, CD31 and UEA1, for mouse and human tissues, respectively, is shown in green (right panels) and cell nuclei stained with DAPI are shown in blue in overlay images (left panels).

Article Snippet: Briefly, ∼1400 RU of recombinant human IGFBP7 (R&D Systems, Minneapolis, MN, USA) and 900 RU of ovalbumin (Sigma) as a reference protein were immobilised on a CM5 sensor chip using the amine-coupling kit supplied by the manufacturer.

Techniques: Immunofluorescence, Staining

Journal: British Journal of Cancer

Article Title: Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

doi: 10.1038/sj.bjc.6605937

Figure Lengend Snippet: In vivo optical imaging of mice bearing orthotopic glioblastoma tumours after intravenous (i.v.) administration of anti-IGFBP7 sdAb labelled with the near-infrared fluorescent probe, Cy5.5. ( A ) Dorsal whole body in vivo optical images of mice bearing U87MG.EGFRvIII brain tumours at indicated time points after i.v. injection of 50 μ g of either anti-IGFBP7 sdAb-Cy5.5 (upper panels), 100 × unlabelled anti-IGFBP7 sdAb followed by anti-IGFBP7 sdAb-Cy5.5 (competitive block, middle panels) or NC sdAb-Cy5.5 (lower panels) (arrows indicate the location of the brain tumour). ( B ) Graph illustrating changes in the average fluorescence concentration determined from an ROI in the brain tumour region in vivo at indicated times after the i.v. injection of sdAbs-Cy5.5. ( C ) Optical images of organs ex vivo and dissected brain tumours 24 h after injection of sdAbs-Cy5.5 ( D ) Graph illustrating the total fluorescence concentration per gram tissue in various organs and dissected tumours imaged ex vivo 24 h after the injection of sdAbs-Cy5.5. In B and D , the data are expressed as mean±s.e.m. for n =5 animals. * Indicates significant difference between anti-IGFBP7 sdAb and both competitively blocked anti-IGFBP7 sdAb and NC sdAb ( P <0.01). # Indicates significant difference between competitively blocked anti-IGFBP7-sdAb and NC sdAb ( P <0.05).

Article Snippet: Briefly, ∼1400 RU of recombinant human IGFBP7 (R&D Systems, Minneapolis, MN, USA) and 900 RU of ovalbumin (Sigma) as a reference protein were immobilised on a CM5 sensor chip using the amine-coupling kit supplied by the manufacturer.

Techniques: In Vivo, Optical Imaging, Injection, Blocking Assay, Fluorescence, Concentration Assay, Ex Vivo

Journal: British Journal of Cancer

Article Title: Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

doi: 10.1038/sj.bjc.6605937

Figure Lengend Snippet: Fluorescent and light microscopic images of mouse orthotopic GBM tumour sections obtained 24 h after intravenous (i.v.) injection of 100 μ g anti-IGFBP7 sdAb-Cy5.5. ( A ) Sections of brain tumour stained with hematoxylin and eosin staining showing brain tumour boundaries from the ipsilatelar healthy brain tissue (left panel, low magnification; right panel, high magnification). Higher magnification fluorescent images of tumour centre ( B ) and contralateral healthy brain region ( C ) showing colocalisation of the systemically injected anti-IGFBP7 sdAb-Cy5.5 (red; middle panels) and brain vessels stained with the 40 μ g fluorescein tomato lectin (green; right panels) injected i.v. 10 min before killing, and their composite overlay images with nuclei stained with DAPI (blue; left panels). Scale bar: 50 μ m. ( D ) Lower magnification image of the tumour and normal brain illustrating that injected anti-IGFBP7 sdAb-Cy5.5 (red) decorates selectively a majority of tumour vessels 24 h after i.v. injection. Scale bar: 100 μ m.

Article Snippet: Briefly, ∼1400 RU of recombinant human IGFBP7 (R&D Systems, Minneapolis, MN, USA) and 900 RU of ovalbumin (Sigma) as a reference protein were immobilised on a CM5 sensor chip using the amine-coupling kit supplied by the manufacturer.

Techniques: Injection, Staining

Journal: British Journal of Cancer

Article Title: Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

doi: 10.1038/sj.bjc.6605937

Figure Lengend Snippet: ( A ) Representative transmission electron microscopy image of NPs at two magnifications. ( B ) Schematic diagram of the anti-IGFBP7 sdAb conjugated to a PEGylated Fe 3 O 4 NP and labelled with Cy5.5. ( C ) Graph of 10 nm core PEGylated NP-Cy5.5 concentration in serum over time, fitted to a two-compartment, IV-bolus model (WinNonLin 5.2).

Article Snippet: Briefly, ∼1400 RU of recombinant human IGFBP7 (R&D Systems, Minneapolis, MN, USA) and 900 RU of ovalbumin (Sigma) as a reference protein were immobilised on a CM5 sensor chip using the amine-coupling kit supplied by the manufacturer.

Techniques: Transmission Assay, Electron Microscopy, Concentration Assay

Journal: British Journal of Cancer

Article Title: Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

doi: 10.1038/sj.bjc.6605937

Figure Lengend Snippet: In vivo optical imaging of the biodistribution of non-targeted and anti-IGFBP7 sdAb-targeted Fe 3 O 4 nanoparticles labelled with Cy5.5 injected at 10 mg Fe kg –1 intravenous (i.v.) in mice bearing orthotopic glioblastoma tumours. ( A ) In vivo images of the head indicated time points after the injection of anti-IGFBP7-targeted (upper panels) and non-targeted (lower panels) NPs. ( B ) In vivo optical images of whole animal body (upper panels) 72 h after i.v. injection of anti-IGFBP7-targeted and non-targeted NPs and corresponding ex vivo organ images after animal killing by perfusion (bottom panels). ( C ) Graph showing changes of the average fluorescence concentration in the brain tumour region in vivo at indicated times after the injection of either anti-IGFBP7 sdAb-targeted or non-targeted-NPs-Cy5.5. ( D ) Graph illustrating the total fluorescence concentration per gram tissue in organs imaged ex vivo 72 h after the injection of either anti-IGFBP7 sdAb-targeted or non-targeted-NPs-Cy5.5. In C and D , data are expressed as mean±s.e.m. for n =5 animals. ** Indicates significant difference between anti-IGFBP7 sdAb-NP-Cy5.5 and non-targeted-NP-Cy5.5 ( P <0.001). * Indicates significant difference between the anti-IGFBP7 sdAb-NP-Cy5.5 and non-targeted-NP-Cy5.5 ( P <0.01).

Article Snippet: Briefly, ∼1400 RU of recombinant human IGFBP7 (R&D Systems, Minneapolis, MN, USA) and 900 RU of ovalbumin (Sigma) as a reference protein were immobilised on a CM5 sensor chip using the amine-coupling kit supplied by the manufacturer.

Techniques: In Vivo, Optical Imaging, Injection, Ex Vivo, Fluorescence, Concentration Assay

Journal: British Journal of Cancer

Article Title: Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

doi: 10.1038/sj.bjc.6605937

Figure Lengend Snippet: Fluorescent microscopic images of mouse GBM tumour sections obtained 72 h after intravenous injection of 10 mg Fe kg –1 of either anti-IGFBP7 sdAb-targeted ( A ) or non-targeted ( B ) Fe 3 O 4 nanoparticles labelled with Cy5.5 (red). ( C ) Contralateral healthy brain region of animals injected with anti-IGFBP7-sdAb-targeted NPs. Mice were also injected with 40 μ g of FITC-labelled tomato lectin, 10 min before killing to stain blood vessels in vivo . Lectin staining (green; right panels) colocalises with the Cy5.5 signal (red; middle panels) of NPs in overlay images (left panels; cell nuclei-blue) only in the tumour region of animals injected with anti-IGFBP7-targeted NPs. Scale bar: 50 μ m.

Article Snippet: Briefly, ∼1400 RU of recombinant human IGFBP7 (R&D Systems, Minneapolis, MN, USA) and 900 RU of ovalbumin (Sigma) as a reference protein were immobilised on a CM5 sensor chip using the amine-coupling kit supplied by the manufacturer.

Techniques: Injection, Staining, In Vivo

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: Correlation between the expression of IGFBP-rP1 immunohistochemical staining and clinicopathological characteristics in colorectal cancer

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: Correlation between the expression of IGFBP-rP1 immunohistochemical staining and EMT markers in colorectal cancer

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: IGFBP-rP1 overexpression blocks EMT in CRC. ( a and b ) Well-established EMT markers were analyzed by western blot after IGFBP-rP1 transfection in SW620 and CW2 cells. E-cadherin expression was markedly induced and the mesenchymal markers N-cadherin, vimentin, and MMP9 were decreased in IGFBP-rP1-transfected cells (-rP1) compared with control vector-transfected (-ctrl) and parental cells (-blk). Equal loading was confirmed by GAPDH. ( c ) SW620 cells were treated with rhIGFBP-rP1 protein (4 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. ( d ) Immunofluorescence images of SW620 cells stained for E-cadherin, β -catenin, and F-actin. The images were taken at × 630 (for E-cadherin and β -catenin ) and × 1000 (for F-actin). Arrow: Lamellipodia and microspike formation. DAPI and β -catenin staining was showed seperately and then the merged images were showed. ( e and f ) Wound-healing and transwell motility assays for SW620-ctrl and SW620-rP1 cells. The motility was drastically decreased in IGFBP-rP1 transfected SW620 cells ( × 100). Cell motility detemined by wound-healing assay was quantified as an inverse ratio of gap distance (GD) at 48 h relative to that at 0 h. * P < 0.05. The data were expressed as mean+S.D. of three independent experiments

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: Over Expression, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Wound Healing Assay

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: IGFBP-rP1 knockdown promotes EMT in SW480 cells. ( a ) The effectiveness of shRNA interference was confirmed by real-time RT-PCR and western blot analyses. The data were expressed as mean+S.D. of three independent experiments. ( b ) The expression of EMT markers was detected by western blot in scrambled control cells (shNC) and two stable IGFBP-rP1 knockdown cell clones (sh2 and sh3). ( c ) Nuclear expression of β -catenin detected by western blot in two SW480/shIGFBP-rP1 cell lines. Relocalization of β -catenin from adherens junctions of the membrane to cytoplasm and nucleus detected by immunofluorescence. ( d ) Wound-healing and transwell motility assays for SW480 cells expressing shRNA directed against IGFBP-rP1 or scrambled control shRNA ( × 100). * P <0.05. The data were expressed as mean+S.D. of three independent experiments. ( e and f ) The addition of rhIGFBP-rP1 protein to stable IGFBP-rP1-knockdown cells. SW480-sh2 cells were treated with rhIGFBP-rP1 protein (1 μg/ml) for 48 h and the expression of EMT markers was detected by western blot. Transwell motility assay was performed in SW480-sh2 cells treated with rhIGFBP-rP1 ( × 100) and value was shown as mean+S.D. of three independent experiments

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: shRNA, Quantitative RT-PCR, Western Blot, Expressing, Clone Assay, Immunofluorescence, Motility Assay

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: Tumorigenicity assay in nude mice. ( a ) SW620-ctrl and SW620-rP1 cells were injected subcutaneously into nude mice ( n =4). Photographs of the tumors and mice were taken at the end of the study. ( b ) IGFBP-rP1 overexpression showed a lower growth rate (Mann–Whitney U -test). Points and bars represent the average±S.D. ( c ) Weights of xenograft tumors with and without IGFBP-rP1 overexpression. ( d ) Photographs of tumors and nude mice after SW480-sh2 and SW480-shNC cells were injected subcutaneously ( n =4). ( e ) IGFBP-rP1 knockdown increased the tumor growth rate in nude mice (Mann–Whitney U -test). Points and bars represent the average±S.D. ( f ) The average tumor was heavier in the SW480-sh2 group than the SW480-shNC group. ( g and h ) EMT markers examined by western blot in harvested mouse tumor samples (GAPDH as a loading control)

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: Tumorigenicity Assay, Injection, Over Expression, MANN-WHITNEY, Western Blot

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: IGFBP-rP1 inhibited tumor metastasis in NOD/SCID mice. ( a ) Representative gross images and hematoxylin and eosin-stained lung sections from animals injected intravenously with control or IGFBP-rP1 overexpressing SW620 cells. Arrows, curve, and triangle: metastatic nodes in the lung. Scale bars, 9 mm (right top) and 8 mm (right bottom). ( b ) Box plot showing the number of lung metastatic nodules. P , by Mann–Whitney U -test. ( c ) Representative images of pleural metastasis of animals injected intravenously with control SW620 cells. ( d ) Quantification of the pleural nodules in each experimental group. P , by Mann–Whitney U -test. ( e ) Representative H&E images of pleural invasion and costal cartilage invasion tissue were shown, respectively. Scale bars, 6 mm (right) and 800 μm (left)

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: Staining, Injection, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: IGFBP-rP1 inhibits TGF- β receptor expression and its downstream signaling in SW620 and SW480 cells. ( a ) SW620 cell lysates from blk, vector, and IGFBP-rP1 cells were analyzed the indicated proteins by western blot. ( b ) SW480 cell lysates from scramble shRNA and IGFBP-rP1 shRNA cells were analyzed the indicated proteins by western blot. ( c ) Western blot of the indicated antibodies in two SW480-IGFBP-rP1 knockdown cell clones and controls untreated or treated with 10 ng/ml SB431542 for 48 h

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: Expressing, Plasmid Preparation, Western Blot, shRNA, Clone Assay

Journal: Cell Death & Disease

Article Title: IGFBP-rP1 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer

doi: 10.1038/cddis.2015.59

Figure Lengend Snippet: IGFBP-rP1 blocks TGF- β 1-induced EMT in HT29 cells. ( a ) HT29 cells were incubated with TGF- β 1 (10 ng/ml) for the indicated times. The levels of P-smad2/3 were assessed by western blot. ( b ) Expression of EMT markers assessed by western blot. ( c ) TGF- β 1 promoted cell migration determined by transwell migration assay. The results were shown as mean+S.D. from triplicate experiments. ( d , e ) Western blot of the indicated antibodies and transwell migration assay in HT29 cells and controls untreated or treated with 1 μg/ml rhIGFBP-rP1 and/or 10 ng/ml TGF- β 1. The results were shown as mean+S.D. from triplicate experiments

Article Snippet: Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA).

Techniques: Incubation, Western Blot, Expressing, Migration, Transwell Migration Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 is downregulated in human HCC samples. A. Analysis of IGFBP7 expression in tissue microarray by immunohistochemistry. C. Fluorescence In Situ Hybridization (FISH) was performed on human HCC samples for IGFBP7 and CEP4 (probe targeting pericentromeric region of chromosome 4). Red: IGFBP7; green: CEP4.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Expressing, Microarray, Immunohistochemistry, Fluorescence, In Situ Hybridization

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 is downregulated in human HCC cell lines and by AEG-1. A. Determination of IGFBP7 mRNA expression by real-time PCR in the indicated cells. THLE-3 is normal immortal human hepatocytes. GAPDH was used as normalization control. B. Secreted IGFBP7 protein level in the conditioned media of the indicated cells determined by ELISA. C. Determination of IGFBP7 mRNA expression by real-time PCR in Hep-pc-4 (pc-4) cells and three independent clones of HepG3 cells overexpressing AEG-1. D. Immunofluorescence detection of IGFBP7 protein in Hep-pc-4 (pc-4) and Hep-AEG-1-14 (AEG1-14) cells. E. Immunohistochemical analysis of AEG-1 and IGFBP7 expression in normal liver and matched HCC from the same patient. The figure represents data from one patient. Similar finding was observed in 13 out of 18 HCC patients. For A-C, data represents mean ± SEM of three independent experiments. *: p<0.05.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Clone Assay, Immunofluorescence, Immunohistochemical staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: Overexpression of IGFBP7 inhibits growth of AEG-1-overexpressing cells. Stable clones of Hep-AEG1-14 cells expressing IGFBP7 (IGFBP7-11 and IGFBP7-12) were generated by selection with zeocin. Zeocin-resistant clone of Hep-AEG1-14 cells (Control-2) served as a control. A. IGFBP7 mRNA expression in the indicated cells detected by real-time PCR. B. Secreted IGFBP7 protein level in the indicated cells detected by ELISA. C. Cell viability (MTT) assay of the indicated cells. D. Colony formation assay of the indicated cells. For A-D, data represents mean ± SEM of three independent experiments. *: p<0.05. E. Western blot analysis performed in the indicated cells with the indicated antibodies. EF1α was used as loading control.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Over Expression, Clone Assay, Expressing, Generated, Selection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, MTT Assay, Colony Assay, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 induces senescence. A. Photomicrograph of Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG-1-14 cells stained for senescence-associated β-galactosidase (SA-β-gal) after 1 week of culture. B. Graphical representation of quantification of SA-β-gal positive cells. At least 1,000 cells were counted for each group. Data represents mean ± SEM of three independent experiments. *: p<0.05. C. Photomicrograph of Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG-1-14 cells stained for γ-H2AX and counterstained with DAPI to stain the nucleus. D. B. Graphical representation of quantification of γ-H2AX foci/cell. At least 100 cells were scored for each group. Data represents mean ± SEM of three independent experiments. *: p<0.05.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Clone Assay, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: Overexpression of IGFBP7 inhibits AEG-1-mediated tumorigenesis in nude mice. Subcutaneous xenografts were established in athymic nude mice using Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG-1-14 cells. A. A representative photograph of tumor-bearing mice at the end of the study. B. Measurement of tumor volume at the indicate time point. Data represents mean ± SEM. *: p<0.05. C. Tumor sections were immunostained for IGFBP7, CD31 and Ki-67.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Over Expression, Clone Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 inhibits angiogenesis. A. Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG1-14 cells were implanted in chicken chorioallantoic membrane (CAM) and neovascularization was photographed. B. Graphical representation of new blood vessel formation in CAM when the indicated cells were implanted. The numbers indicate percentage of new blood vessels arising from the existing blood vessels in naïve CAM when VEGF-treated CAM was considered as 100%. Data represents mean ± SEM. *: p<0.05. C. HUVECs were treated with conditioned media from the indicated cells and tube formation was photographed. D. Graphical representation of tube formation by HUVEC treated with conditioned media from the indicated cells. The numbers indicate percentage of tube-like structures when VEGF-treated tube formation was considered as 100%. The data represents mean ± SEM. *: p<0.05.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Clone Assay