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

Journal: International Journal of Oncology

Article Title: Cancer-associated fibroblast-induced M2-polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor-1 pathway

doi: 10.3892/ijo.2021.5239

Figure Lengend Snippet: PAI-1 is the key factor secreted by TAMs following CAF-CM stimulation and promotes tumor malignant behavior in vitro . (A) Original image and (B) spot pixel value from a cytokine array revealed the profiles of paracrine factors in the M0-CM, TAM (Ca)-CM and TAM(CAF)-CM. (C) Western blot and (D) ELISA results indicated that PAI-1 was upregulated in the TAM(CAF) group compared with that in the M0 and TAM(Ca) groups. (E) Cell Counting Kit-8 and (F) colony formation assays showed that the inhibition of PAI-1 in TAM(CAF)-CM suppressed the enhanced proliferation of Huh-7 cells. (G) Wound healing (scale bar, 400 µ m) and (H) Transwell and (I) Matrigel (scale bar, 200 µ m) assays indicated that the inhibition of PAI-1 in the TAM(CAF)-CM group suppressed the enhanced migration and invasion of the Huh-7 cells. * P<0.05; ** P<0.01. CAF, cancer-associated fibroblast; CM, conditioned medium; CXCL12, C-X-C motif chemokine ligand 12; CXCL5, C-X-C motif chemokine ligand 5; PAI-1, plasminogen activator inhibitor-1; PDGF-AA, platelet derived growth factor-AA; TAM, tumor-associated macrophage; TIM-3, T-cell immunoglobulin mucin 3; M0, macrophages; NS, not significant.

Article Snippet: The Human IL-6 Quantikine ELISA kit (cat. no. D6050), Human Serpin E1/PAI-1 Quantikine ELISA kit (cat. no. DSE100) and Human CXCL12/stromal cell-derived factor 1α Quantikine ELISA kit (cat. no. DSA00) were purchased from R&D Systems Inc., to detect the concentrations of IL-6, PAI-1 and CXCL12 in the CM, according to the manufacturer's instructions.

Techniques: In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Inhibition, Migration, Derivative Assay

Journal: International Journal of Oncology

Article Title: Cancer-associated fibroblast-induced M2-polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor-1 pathway

doi: 10.3892/ijo.2021.5239

Figure Lengend Snippet: CAF-derived CXCL12 induces the secretion of PAI-1 in TAM(CAF). (A) Original image and (B) spot pixel value from a cytokine array to identify the different patterns of molecules in cancer-CM and CAFs-CM. CXCL12 gene expression and its secretion were increased in CAFs compared with that in the Huh-7 and Lx-2 cells following (C) RT-qPCR and (D) ELISA. (E) CXCR4 gene expression in M0, TAM(Ca) and TAM(CAF) was analyzed using RT-qPCR. (F) RT-qPCR and (G) ELISA results demonstrated that the gene expression level and secretion of PAI-1 were decreased in TAM(CAF) after CXCL12 neutralization in CAF-CM, respectively. (H) Schematic representation of the proposed interactions between HCC, TAMs and CAFs. * P<0.05; ** P<0.01. CAF, cancer-associated fibroblast; CM, conditioned medium; CCL5, C-C motif chemokine ligand 5; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C Motif chemokine receptor 4; HCC, hepatocellular carcinoma; HSCs, hepatic stellate cells; PAI-1, plasminogen activator inhibitor-1; PDGF-AA, platelet derived growth factor-AA; RT-qPCR, reverse transcription-quantitative PCR; TAM, tumor-associated macrophage; Ab, antibody.

Article Snippet: The Human IL-6 Quantikine ELISA kit (cat. no. D6050), Human Serpin E1/PAI-1 Quantikine ELISA kit (cat. no. DSE100) and Human CXCL12/stromal cell-derived factor 1α Quantikine ELISA kit (cat. no. DSA00) were purchased from R&D Systems Inc., to detect the concentrations of IL-6, PAI-1 and CXCL12 in the CM, according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Neutralization, Reverse Transcription, Real-time Polymerase Chain Reaction