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