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

Journal: Immunology

Article Title: Novel role of regulatory T cells in limiting early neutrophil responses in skin

doi: 10.1111/j.1365-2567.2010.03333.x

Figure Lengend Snippet: Depletion of CD25+ cells promotes neutrophil accumulation at the site of tumour inoculation. Mice were treated with either isotype control monoclonal antibodies (GL113; a) or CD25-specific monoclonal antibodies (PC61; b) and injected 1 day later with 105 B16Fas ligand. At 24 hr post-injection, mice were killed and the skin surrounding the injection site was collected for histology. Interleukin-8 receptor (IL-8R) -stained 5-μm paraffin-mounted sections were generated to identify neutrophils. Areas of neutrophil infiltration are enclosed by the blue lines. Sections are representative of at least five mice per group at 24 hr. (c) Examples of neutrophils (indicated by arrows) which stain dark brown with multi-lobed nuclei. (d) The numbers of neutrophils, assessed as polymorphonuclear cells with IL-8R expression, are given where each symbol represents the score for one mouse. Statistical significance was evaluated by Mann–Whitney U-test (*P < 0·05). (e) The relative expression of CXCL1 and CXCL2, assessed by real-time PCR, in the skin of GL113-treated and PC61-treated B16FasL-inoculated mice.

Article Snippet: Neutrophils were detected using rabbit anti-mouse interleukin-8 receptor B (IL-8RB; K-19; Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation with biotinylated swine anti-rabbit abs (Dako, Glostrup, Denmark).

Techniques: Injection, Staining, Generated, Expressing, MANN-WHITNEY, Real-time Polymerase Chain Reaction