Journal: International Journal of Molecular Sciences

Article Title: Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

doi: 10.3390/ijms14059751

Figure Lengend Snippet: VEGF expression and production in ovarian epithelial carcinoma cells was induced by resistin. ( A ) Time course of 100 ng/mL resistin treatment on VEGF mRNA levels in HO-8910 cells; ( B ) Dose-response effect of 24-h resistin treatment on VEGF mRNA levels in HO-8910 cells; ( C ) Attenuation of resistin-induced VEGF expression by anti-resistin neutralizing antibody in HO-8910 cells. Cells were pretreated with or without anti-resistin neutralizing antibody (1:1000 or 1:250) for 1 h, then incubated in the presence of resistin 100 ng/mL for 24 h. Relative mRNA levels were normalized to that of untreated cells; ( D ) Dose-response effect of 24-h resistin treatment on VEGF peptide levels in the medium of HO-8910 cells; ( E ) Effects of resistin on VEGF promoter (pLuc 1) activity in HO-8910 cells. Cells were transfected with pLuc 1 together with control plasmid with use of jetPEI reagent. After transfection for 24 h, cells were stimulated with 100 ng/mL resistin or PBS, then luciferase activity was measured. Relative luciferase activity was normalized with respect to the activity in untreated cells. The results are representative of four independent experiments performed in triplicate. Data are means ± SEM. * p < 0.05; ** p < 0.01 vs. untreated cells; # p < 0.05, ## p < 0.01 vs. resistin treatment alone.

Article Snippet: Anti-human VEGF neutralizing antibody was purchased from Oncogene Science (Cambridge, MA, USA).

Techniques: Expressing, Incubation, Activity Assay, Transfection, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

doi: 10.3390/ijms14059751

Figure Lengend Snippet: PI3K/Akt pathway was activated upon resistin stimulation. ( A ) Effects of resistin on PI3K/Akt pathway activation in HO-8910 cells. Cells were incubated with 100 ng/mL resistin for 1 h and whole-cell lysates underwent Western blotting to detect the phosphorylation of p85 subunit of PI3K and Akt (Ser 473); ( B – D ) Attenuation of resistin-induced VEGF mRNA expression ( B ), peptide secretion ( C ) and promoter activity ( D ) by LY294002 and wortmannin in HO-8910 cells. Cells were pretreated with (+) or without (−) LY294002 (20 μM) and wortmannin (2 μM) for 1 h, then incubated in the presence (+) or in the absence (−) of resistin 100 ng/mL for 24 h. Relative VEGF mRNA levels were normalized with respect to the levels for untreated cells. The basal promoter activity of each test plasmid is indicated as luciferase activity normalized by each internal control activity (PRL-TK). Relative luciferase activity was normalized with respect to the activity in untreated cells. The results are representative of four independent experiments performed in triplicate. Data are means ± SEM. ** p < 0.01 vs. untreated cells, ## p < 0.01 vs. resistin treatment alone.

Article Snippet: Anti-human VEGF neutralizing antibody was purchased from Oncogene Science (Cambridge, MA, USA).

Techniques: Activation Assay, Incubation, Western Blot, Phospho-proteomics, Expressing, Activity Assay, Plasmid Preparation, Luciferase, Control

Journal: International Journal of Molecular Sciences

Article Title: Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

doi: 10.3390/ijms14059751

Figure Lengend Snippet: Sp1 acted on the motif of VEGF promoter. ( A ) Effects of resistin on Sp1 activation in HO-8910 cells. Cells were incubated with 100 ng/mL resistin for 2 h and whole-cell lysates underwent Western blotting to detect the phosphorylation of Sp1 at either Thr-453 or Thr-739; ( B ) Scheme of the series of plasmid constructs containing dissected fragments of VEGF promoter; ( C ) Relative luciferase activity in transient transfection assays using the series of plasmid constructs (schematically shown in B ) and pSp1-luciferase reporter. Relative luciferase activity was normalized with respect to the activity of PGL3-basic in untreated cells. Data are mean ± SEM from four independent triplicated experiments; ( D ) Chromatin immunoprecipitation of VEGF promoter complexes. HO-8910 cells underwent immunoprecipitation with anti-Sp1 antibody or control IgG. Immunoprecipitated chromatin fragments were amplified by PCR with specific primers targeting the predicted Sp1 binding motifs within the VEGF promoter; ( E ) Effects of various competitors in EMSA of resistin-stimulated HO-8910 cells. Competitors, including Sp1, AP-2 and Egr-1 consensus sequences, were added to the reaction mixture at a 100-fold molar excess to labeled probe, the VEGF promoter fragment containing predicted Sp1 binding motifs. Lane 2 contained no competitor. Nuclear extracts were prepared from HO-8910 cells and underwent EMSA. Cells were pre-incubated with 100 ng/mL resistin for 24 h.

Article Snippet: Anti-human VEGF neutralizing antibody was purchased from Oncogene Science (Cambridge, MA, USA).

Techniques: Activation Assay, Incubation, Western Blot, Phospho-proteomics, Plasmid Preparation, Construct, Luciferase, Activity Assay, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Control, Amplification, Binding Assay, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

doi: 10.3390/ijms14059751

Figure Lengend Snippet: HO-8910 cells-derived VEGF mediated resistin-induced angiogenesis of EA.hy926 cells. ( A ) Capillary-like tube formation assay of EA.hy926 cells co-cultured with HO-8910 cells under resistin (100 ng/mL) stimulation for 18 h. The total length of capillary-like tubes was measured and normalized with resistin-untreated EA.hy926 cells. Data are mean ± SEM from three independent experiments. ** p < 0.01 vs. resistin-untreated EA.hy926 cells only, ## P < 0.01 vs. resistin-treated EA.hy926 cells; ( B ) Blockage of VEGF by neutralizing antibody retarded capillary-like tube formation ability of EA.hy926 cells co-cultured with resistin-stimulated HO-8910 cells. The total length of capillary-like tubes was measured and normalized with controls. Data are mean ± SEM from three independent experiments. ** p < 0.01 vs. untreated cells, ## p < 0.01 vs. resistin treatment alone; ( C ) Representative microscopic view of capillary-like tube formation in EA.hy926 cells co-cultured with HO-8910 cells under resistin (100 ng/mL) stimulation without or with VEGF neutralizing antibody treatment.

Article Snippet: Anti-human VEGF neutralizing antibody was purchased from Oncogene Science (Cambridge, MA, USA).

Techniques: Derivative Assay, Capillary Tube Formation Assay, Cell Culture