Journal: The Journal of Biological Chemistry

Article Title: Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis *

doi: 10.1074/jbc.M115.664037

Figure Lengend Snippet: Microglial secretome analysis by SILAC. A, schematic representation of the work flow used to study the variations in the CHME-3 secretome induced by CM of U87 silenced or not for MCSF using SILAC. B, volcano plot representation of SILAC results. The x axis shows the log 2 H/M ratio derived by taking the ratio of mean intensity of heavy label to mean intensity of medium label for each protein. The y axis shows the p value expressed in −log10 scale. A total of 67 proteins were significantly differentially regulated, of which 28 were up-regulated and 39 were down-regulated. Each dot represents one protein in the plot. Red and green dots, up- and down-regulation, respectively. The location of VEGFA and IGFBP1 is indicated. C, annotated MS/MS spectra of five different IGFBP1 peptides.

Article Snippet: The following reagents were used in this study: recombinant MCSF (Biolegend), MCSF, SYK- and IGFBP1-specific siRNA (Dharmacon), MCSFR inhibitor GW2580 (LC Laboratories), Bay 11-7082 (Sigma-Aldrich), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 , U0126 and Bay 61-3606 (Calbiochem), anti-AKT and anti-phospho-AKT (Cell Signaling, 4691 and 4060, respectively), anti-IGFBP1 (R&D Systems, MAB675), anti-MCSFR (Abcam, ab89907), anti-MCSF (Novus Biologicals, NB110-57176), anti-CD68 (Biogenex, MU416-UC), anti-CD86 (Epitomics, 1858-1), anti-CD204(Sigma-Aldrich, HPA000272), MCSF and IGFBP1 ELISA kit (R&D Systems; DY216 and DY871, respectively), and luciferase assay reagent (Promega).

Techniques: Multiplex sample analysis, Derivative Assay, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis *

doi: 10.1074/jbc.M115.664037

Figure Lengend Snippet: Microglial IGFBP1 is a mediator of MCSF-induced angiogenesis. A and C, VEGFA protein and transcript levels in CHME-3 measured by SILAC and qRT-PCR methods, respectively. The expression levels of VEGFA in CHME-3 upon treatment with CM from MCSF-silenced U87 were normalized to levels present in CHME-3, which was treated with CM from siNT-transfected (control) U87. B and D, IGFBP1 protein and transcript levels in CHME-3 measured by SILAC and qRT-PCR methods, respectively. The expression levels of IGFBP1 in CHME-3 upon treatment with CM from MCSF-silenced U87 were normalized to levels present in CHME-3 that was treated with CM from siNT-transfected (control) U87. E and F, IGFBP1 levels, measured by ELISA, in CHME-3 cell supernatant with different CM treatment as indicated. The IGFBP1 levels were expressed as a percentage of the levels measured in the supernatant of the CHME-3 cells, which was treated with CM from corresponding siNT-transfected glioma cells. A t test was performed, and the p values are indicated. G and H, IGFBP1 transcript and protein levels in the CHME-3 upon transfection with either siNT or siIGFBP1, as measured by qRT-PCR (48 h) and ELISA (72 h) methods, respectively. The expression was normalized to siNT samples. I and J, tube formation assay in various conditions as indicated. I, supernatant from CHME-3 cells in the presence of IgG antibody (CHME-3 sup./IgG Ab); supernatant from CHME-3 cells treated with U251 CM derived from non-targeting siRNA-transfected cells in the presence of IgG antibody (U251 siNT CM/CHME-3 sup./IgG Ab); supernatant from CHME-3 cells treated with U251 CM derived from non-targeting siRNA transfected cells in presence of IGFBP1 antibody (U251 siNT CM/CHME-3 sup./IGFBP1 Ab); and supernatant from CHME-3 cells treated with U251 CM derived from MCSF-silenced cells in the presence of IgG antibody (U251 siMCSF CM/CHME-3 sup./IgG Ab). J, supernatant from CHME-3 cells transfected with siNT and subsequently treated with U251 CM (U251 CM/CHME-3 siNT sup.) and supernatant from CHME-3 cells transfected with siIGFBP1 and subsequently treated with U251 CM (U251 CM/CHME-3 siIGFBP1 sup.). A t test was performed, and the p values are indicated. K, scatter plot representation of IGFBP1 transcript levels using the TCGA data set, which consisted of normal (n = 10) and GBM (n = 572) samples. A non-parametric t test was performed, and the p value is indicated. The horizontal line represents the mean value. ns, non-significant. Error bars, S.D.

Article Snippet: The following reagents were used in this study: recombinant MCSF (Biolegend), MCSF, SYK- and IGFBP1-specific siRNA (Dharmacon), MCSFR inhibitor GW2580 (LC Laboratories), Bay 11-7082 (Sigma-Aldrich), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 , U0126 and Bay 61-3606 (Calbiochem), anti-AKT and anti-phospho-AKT (Cell Signaling, 4691 and 4060, respectively), anti-IGFBP1 (R&D Systems, MAB675), anti-MCSFR (Abcam, ab89907), anti-MCSF (Novus Biologicals, NB110-57176), anti-CD68 (Biogenex, MU416-UC), anti-CD86 (Epitomics, 1858-1), anti-CD204(Sigma-Aldrich, HPA000272), MCSF and IGFBP1 ELISA kit (R&D Systems; DY216 and DY871, respectively), and luciferase assay reagent (Promega).

Techniques: Multiplex sample analysis, Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: A mutation in NLRC4 induces overproduction of IL-1β through activation of caspase-1. (a) Flag-tagged wild-type, mutant NLRC4 expression vectors or empty vector (EV) were transfected into 293T cells. 2 d later, after the cell lysates were subjected to SDS-PAGE, Western blotting was performed with an anti-Flag or anti-actin antibody. Red triangle indicates NLRC4. The data in the figure are representative of three independent experiments. (b) Cell lysates from 293T cells transfected with empty vector (EV), flag-tagged wild-type, or mutant NLRC4 were subjected to Blue Native PAGE and the expression of the tagged proteins or actin was evaluated by Western blotting with an anti-Flag or actin antibody, respectively. The data in the figure are representative of three independent experiments. (c) 293T cells were transfected with a CASP1 expression vector together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the expression of procaspase-1, cleaved active caspase-1 (10 kD), and actin was evaluated by Western blotting. Red triangles indicate procaspase-1, cleaved active caspase-1, and actin. The data in the figure are representative of five independent experiments. (d) 293T cells were transfected with expression vectors for CASP1 and IL1B together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the concentration of IL-1β in the supernatant was evaluated by ELISA. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (e) Peripheral blood mononuclear cells were transfected with three concentrations of Prgl and cultured for 48 h. The concentrations of IL-1β in the supernatants were measured by ELISA. The data shown are means ± SD (**, P < 0.01). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Activation Assay, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, Blue Native PAGE, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: BM-derived macrophages that overexpress mutant Nlrc4 secrete IL-1β. BM-derived macrophages (BMMC) infected with control retrovirus, retroviruses encoding wild-type, or mutant Nlrc4 were stimulated with 10 µg/ml LPS for 1 d in the absence (black) or presence (gray) of a caspase-1 inhibitor. As the control, unstimulated BMMCs were used (white). The concentrations of IL-1β and IL-6 in the supernatant were measured by ELISA. The data shown are means ± SD (*, P < 0.05; n = 5). The data shown in this figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Derivative Assay, Mutagenesis, Infection, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cytokine production in mu-Nlrc4 mice or mice reconstituted with mutant Nlrc4 transduced BM cells. (a) Splenocytes from wild-type or mu-Nlrc4 mice were stimulated with LPS for 24 h, and the concentration of IL-1β in each supernatant was evaluated by ELISA. The data shown are means ± SD (**, P < 0.01). N.D.: not detected. The data in the figure are representative of three independent experiments. (b) The concentrations of IL-1β, IL-17A, and G-CSF in the serum of wild-type (white) and mu-Nlrc4 (black) mice at the age of 8 wk were evaluated by ELISA. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of three independent experiments. (c) Intraperitoneal cells or splenocytes from C57BL/6 mice transplanted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus were evaluated for the number of Gr1 + CD11b + cells 2 mo after the transplantation. The data shown are means ± SD (*, P < 0.05; n = 5 in each group). The data in the figure are representative of three independent experiments. (d) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. Serum IL-1β, IL-17A, and G-CSF were evaluated by ELISA 6 wk after BM transplantation. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Control, Virus, Transplantation Assay, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cold exposure of mu-Nlrc4 mice increases autoinflammation. (a) The footpads of C57BL/6 (wild type; white) and mu-Nlrc4 (black) mice were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (b) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. The footpads were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (c) The entire bodies of control C57BL/6 and mu-Nlrc4 mice were exposed to 4°C for 1 min and kept for 3 min at room temperature, and then the serum concentrations of IL-1β were measured. The data are shown as means ± SD. N.D., not detected. (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (d) MC/9 cells infected with control retrovirus (white), retroviruses encoding wild-type (black), or mutant Nlrc4 (gray) were cultured at 32 or 37°C for 48 h. 293T cells were transfected with control vector (white), vectors encoding wild-type (black), or mutant NLRC4 (gray) and 48 h later, cells were washed and cultured at 32 or 37°C for 6 h. The levels of IL-1β in the supernatants were evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (*, P < 0.05). The data in the figure are representative of three independent experiments. (e) Peripheral mononuclear cells from an FCAS patient or a healthy control were cultured at 32°C for 48 h in the presence (black) or absence (white) of a caspase inhibitor or cultured at 37°C for 48 h. The level of IL-1β in the supernatant was evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (**, P < 0.01). The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Irradiation, Infection, Control, Virus, Mutagenesis, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: The accumulation of neutrophils in mu-Nlrc4 mice depends on IL-1β and IL-17A. (a) Spleen cells from C57BL/6 or mu-Nlrc4 mice at the age of 12 wk were stimulated with 25 ng/ml PMA and 1 µg/ml ionomycin in the presence of 2 µM monensin for 5 h and then stained with anti–TCR-β, anti–TCR-γδ, anti-B220, anti-CD11c, anti-NK1.1, anti-Gr1, and anti-CD11b antibodies. The cells were fixed and stained with anti–IL-17A antibody. The expression of IL-17A was analyzed by flow cytometry. The number in each rectangle indicates the percentage of IL-17A–positive cells in the lineage-negative fraction. The data in the figure are representative of five independent experiments. (b) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti-CD4, anti-CD8, anti-Thy1.2, anti-Gr1, or anti–IL-1β antibody five times at 3-d intervals. The serum IL-17A levels 2 d after final antibody treatment was measured by ELISA. Control sera from C57BL/6 (wild type) mice was used. The data shown are means ± SD (**, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments. (c) mu-Nlrc4 mice at the age of 12 wk were injected twice with control rat IgG, anti-CD4, anti-CD8, or anti-Thy1.2 at 3 d intervals. The spleen cells from mice 1 d after the final injection were stained with anti-CD4 and anti–TCR-β (for anti-CD8–treated mice), anti-CD8 and anti–TCR-β (for anti-CD4–treated mice), or anti-CD4, anti-CD8, and anti–TCR-β antibodies (for anti-Thy1.2–treated mice). The data in the figure are representative of two independent experiments. (d) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti–IL-1β, anti–IL-17A, or anti–IL-1β, and anti–IL-17A antibody five times at 3-d intervals. The number of Gr1 + CD11b + cells in the spleen 2 d after the final antibody treatment was evaluated by flow cytometry. The data shown are means ± SD (*, P < 0.05; **, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Staining, Expressing, Flow Cytometry, Injection, Control, Enzyme-linked Immunosorbent Assay