Journal: Breast Cancer Research : BCR

Article Title: Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis

doi: 10.1186/bcr3053

Figure Lengend Snippet: Genes differentially regulated by IRF5 in MDA-MB-231 cells.

Article Snippet: Briefly, 100 ng/ml human recombinant CXCL12/SDF-1 (R&D Systems, Minneapolis, MN, USA) was added to 600 μl of phenol red-free DMEM medium supplemented with 10% FBS in the lower chamber.

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis

doi: 10.1186/bcr3053

Figure Lengend Snippet: IRF5 reduces CXCR4 cell surface expression and SDF-1/CXCL12-dependent chemotaxis of MDA-MB-231 cells . A . CXCR4 expression (grey line) in unstimulated cells, shown superimposed on the isotype control (grey shaded area), and CXCR4 expression (black line) after stimulation, was measured by flow cytometry. MDA-MB-231 cells (pBabe and pBIRF5) were treated with the CXCR4 ligand SDF-1 for six hours and CXCR4 expression measured. IRF5 expressing cells show no significant expression of CXCR4. M1, Marker 1. Representative histogram plots from three independent experiments performed in duplicate are shown. B . Cells overexpressing IRF5 are incapable of SDF-1-induced migration when compared to empty vector (EV pBabe) control cells. Data are expressed as mean ± SD of three independent experiments performed in duplicate. Statistical significance was determined by comparing the difference in number of cells migrated between pBabe and pBIRF5 cells; * denotes P < 0.02, ** P < 0.005. C . CXCR4 promoter reporter activity was analyzed by Dual Luciferase assay. MDA-231-pBabe and MDA-231-pBIRF5 were transfected with pGL3 empty vector or pGL3 CXCR4 5'Δ3 promoter and mock-treated with PBS or 100 ng/ml CXCL12. Data are expressed as the mean relative stimulation ± SD from three independent experiments performed in triplicate. Statistical significance was determined by comparing the difference in promoter activity between pBabe and pBIRF5 expressing cells; * denotes P < 0.05.

Article Snippet: Briefly, 100 ng/ml human recombinant CXCL12/SDF-1 (R&D Systems, Minneapolis, MN, USA) was added to 600 μl of phenol red-free DMEM medium supplemented with 10% FBS in the lower chamber.

Techniques: Expressing, Chemotaxis Assay, Control, Flow Cytometry, Marker, Migration, Plasmid Preparation, Activity Assay, Luciferase, Transfection