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: PLoS ONE

Article Title: The Impact of MicroRNA-223-3p on IL-17 Receptor D Expression in Synovial Cells

doi: 10.1371/journal.pone.0169702

Figure Lengend Snippet: Primer for real-time PCR.

Article Snippet: After blocking nonspecific binding with 10% normal rabbit serum in PBS, the sections were incubated with rabbit anti-mouse IL-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse TNF-α antibody (Santa Cruz), rabbit anti-mouse IL-17 antibody (Santa Cruz), or rabbit anti-mouse IL-17RD antibody (LifeSpan Biosciences, Inc. Seattle, WA, USA) at appropriate dilutions for 1 h at room temperature, washed, incubated with biotinylated goat anti-rabbit IgG, washed again and incubated with avidin-biotinylated horseradish peroxidase complex (ABC) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) (VECTASTAIN Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) and counterstained with Mayer's hematoxylin.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: The Impact of MicroRNA-223-3p on IL-17 Receptor D Expression in Synovial Cells

doi: 10.1371/journal.pone.0169702

Figure Lengend Snippet: (A) Results of luciferase assay. Reporter plasmid and mouse pmG/mIl17rd were transfected into HeLa cells with either miR-223-3p overexpression vector (pBA/miR-223-3p) or negative control vector (pBA/NC). Results were expressed as the relative luciferase activity following transfection of the negative control vector. **p < 0.01. (B) Expression of miR-223-3p in miR-223-3p overexpression NIH3T3 cells. Results were expressed as the relative fold change following transfection of cells with the negative control vector. ***p < 0.001. (C) Expression of Il17rd mRNA in miR-223-3p overexpression NIH3T3 cells. Results were expressed as the relative fold change following transfection with the negative control vector. ***p < 0.001. (D) Results of western blot analysis. (E) Relative expression of IL-17RD protein. IL17RD expression was normalized to that of β-actin in the same sample, and results were expressed as a percentage of the expression following transfection with the negative control vector. **p < 0.01. (F) Expression of Il6 mRNA in miR-223-3p overexpression NIH3T3 cells. Results were expressed as the relative fold change following transfection of cells with the negative control vector. ***p < 0.001. All results are presented as the means ± S.E. for each group (n = 3).

Article Snippet: After blocking nonspecific binding with 10% normal rabbit serum in PBS, the sections were incubated with rabbit anti-mouse IL-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse TNF-α antibody (Santa Cruz), rabbit anti-mouse IL-17 antibody (Santa Cruz), or rabbit anti-mouse IL-17RD antibody (LifeSpan Biosciences, Inc. Seattle, WA, USA) at appropriate dilutions for 1 h at room temperature, washed, incubated with biotinylated goat anti-rabbit IgG, washed again and incubated with avidin-biotinylated horseradish peroxidase complex (ABC) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) (VECTASTAIN Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) and counterstained with Mayer's hematoxylin.

Techniques: Luciferase, Plasmid Preparation, Transfection, Over Expression, Negative Control, Activity Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: The Impact of MicroRNA-223-3p on IL-17 Receptor D Expression in Synovial Cells

doi: 10.1371/journal.pone.0169702

Figure Lengend Snippet: (A) Schematic diagram of the luciferase reporter plasmids including IL-17RD3’UTR wild type (WT) or deleted (del) miR-223-3p binding sequences in the 3’UTR in which six nucleotide forming the seed region of miR-223-3p were deleted. (B) Results of luciferase assay. Either pmG/ hIl17rd WT or pmG /hIl17rd del was transfected into HeLa cells with either miR-223-3p overexpression vector (pBA/miR-223-3p) or negative control vector (pBA/NC). Results were expressed as the relative luciferase activity following transfection of cells with the negative control vector. *p<0.05 as compared with the pBA/NC group, #p<0.05 as compared with the pmG/ hIL17rd WT group. (C) Results of miR-223-3p expression in MH7A cells. Results were expressed as the relative fold change following transfection of cells with the negative control vector. ***p < 0.001. (D) Results of Il17rd mRNA expression in MH7A cells. Results were expressed as the relative fold change following transfection of the negative control vector; ***p < 0.001. (E) Results of western blot analysis. (F) Relative expression of IL-17RD protein. IL17RD expression was normalized to that of β-actin in the same sample, and results were expressed as a percentage of the expression following transfection with the negative control vector. *p < 0.05. (G) Results of Il6 mRNA expression in MH7A cells. Results were expressed as the relative fold change following transfection with the negative control vector. ***p < 0.001. (H) Effect of miR-223-3p inhibitor on Il17rd mRNA expression. Results were expressed as the relative fold change with the negative control (NC). *p < 0.05. All results are presented as the means ± S.E. for each group (n = 3).

Article Snippet: After blocking nonspecific binding with 10% normal rabbit serum in PBS, the sections were incubated with rabbit anti-mouse IL-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse TNF-α antibody (Santa Cruz), rabbit anti-mouse IL-17 antibody (Santa Cruz), or rabbit anti-mouse IL-17RD antibody (LifeSpan Biosciences, Inc. Seattle, WA, USA) at appropriate dilutions for 1 h at room temperature, washed, incubated with biotinylated goat anti-rabbit IgG, washed again and incubated with avidin-biotinylated horseradish peroxidase complex (ABC) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) (VECTASTAIN Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) and counterstained with Mayer's hematoxylin.

Techniques: Luciferase, Binding Assay, Transfection, Over Expression, Plasmid Preparation, Negative Control, Activity Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: The Impact of MicroRNA-223-3p on IL-17 Receptor D Expression in Synovial Cells

doi: 10.1371/journal.pone.0169702

Figure Lengend Snippet: The expression of IL-17RD in synovial cells by SKG mice with mild arthritis (arthritis score:0.5) (A) was higher than that by SKG mice with severe arthritis (arthritis score:2.5) (B). The expression of IL-6 in synovial tissues from SKG mice with mild arthritis (C) was lower than that from SKG mice with severe arthritis (D).The expression of IL-17RD in synovial tissues from RA patients with mild arthritis (stage 2) (E) was higher than that from RA patients with severe arthritis (stage 4) (F). Original magnification, ×200.

Article Snippet: After blocking nonspecific binding with 10% normal rabbit serum in PBS, the sections were incubated with rabbit anti-mouse IL-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-mouse TNF-α antibody (Santa Cruz), rabbit anti-mouse IL-17 antibody (Santa Cruz), or rabbit anti-mouse IL-17RD antibody (LifeSpan Biosciences, Inc. Seattle, WA, USA) at appropriate dilutions for 1 h at room temperature, washed, incubated with biotinylated goat anti-rabbit IgG, washed again and incubated with avidin-biotinylated horseradish peroxidase complex (ABC) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) (VECTASTAIN Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) and counterstained with Mayer's hematoxylin.

Techniques: Expressing