Journal: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: A , Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin ( B ) and macrophages ( C ), CD19 ( E ), and myeloperoxidase (MPO) of the AAA wall. B , Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C , Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D , CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E , CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F , MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P , Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P , Macrophage: green, CD11b ( G )/LYVE-1 ( H )/VEGF-C ( I )/MMP-9 ( J )/TGF-β1 ( K )/IL-4 ( L )/IL-8 ( M )/MIP-1α ( N )/IFN-γ ( O )/MCP-1 ( P ): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. Q , Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. R , Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. S , Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm ( A–F ) and 10 µm ( G–S ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: ( A ) Relative IL-8 mRNA expression in M2 macrophages treated with leptin with or without the inhibitors. M2 macrophages were pretreated with JAK inhibitor AG490 (50 μmol/L), PI3K inhibitor LY294002 (10 μmol/L), ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative IL-8 mRNA levels were analyzed by qRT-PCR. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01. ( B ) A representative image of Western blot for IL-8 protein expression in M2 macrophages treated in the same manner as (A). ( C ) A representative image of Western blot showing the time course of leptin-induced p38 and ERK 1/2 phosphorylation. M2 macrophages were treated with leptin (100 ng/mL) for 0–24 hours. ( D ) A representative image of Western blot showing leptin-induced ObR-dependent phosphorylation of ERK 1/2 and p38 and production of IL-8. M2 macrophages were pretreated with a polyclonal anti-human ObR antibody (4 μg/mL) for 16 hours and then treated with PBS or leptin (100 ng/mL). ( E ) Luciferase reporter assay to measure leptin-mediated IL-8 promoter activation. M2 macrophages were pretreated with ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative luciferase units (RLU) normalized to β-galactosidase activity are shown. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Luciferase, Reporter Assay, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: The mouse xenograft model and leptin injection were conducted as in Figure . In the leptin-treated group, polyclonal rabbit anti-mouse IL-8 neutralization antibody (Abbexa Ltd, Cambridge, UK) was injected intraperitoneal in the mice at an initial dose of 0.2 mL per mouse and then 0.1 mL per mouse twice per week for 2 weeks. Polyclonal rabbit IgG (Bioss Ltd, Beijing, China) was used as the control. Mice received the injection of leptin and the antibodies for 2 weeks. ( A ) Tumor volume in the PBS, leptin, leptin + anti-IgG, and leptin + anti-IL8 groups. * P < 0.05. ( B ) Photos of tumor xenografts at day 21 after transplantation of human breast cancer cells. ( C ) Tumor wet weight at day 21 after the cell transplantation. ** P < 0.01. ( D ) Mouse survival. * P < 0.05. ( E ) Representative images of H & E staining of the lung tissue and the figure showing the number of metastatic nodules in the lung. ** P < 0.01. ( F ) Representative images of H & E staining of the liver tissue. ( G ) Representative images of IHC staining of CD68, Ki-67, and IL-8 in the tumor tissue and the figure showing quantitative analysis of the staining. ** P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Injection, Neutralization, Control, Transplantation Assay, Staining, Immunohistochemistry