anti cxcl5  (R&D Systems)

Journal: Scientific reports

Article Title: ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

doi: 10.1038/s41598-022-19290-6

Figure Lengend Snippet: Figure 3. Secreted CXCL5 in the conditioned medium of the co-culture model with resistin-stimulated ADSCs promoted malignant behaviors of breast cancer cells. The isolated ADSCs were treated with resistin at 0 and 100 ng/ml (R0 and R100, respectively) for 48 h, followed by co-culture with MDA-MB-231 cells in the transwell model for another 72 h before the analyses in (A,B,F,G). (A) The conditioned medium from the co-culture of R-ADSCs (R100) or control ADSCs (R0) with MDA-MB-231 cells was collected and analyzed by cytokine/ chemokine proteome array. A total of 102 proteins were detected with duplicates for each protein. The position of CXCL5 on the array was highlighted. (B) Secreted protein level of CXCL5 in the conditioned medium from the co-culture of R-ADSCs (R100) or control ADSCs (R0) with MDA-MB-231 cells was analyzed by ELISA. (C) MDA-MB-231 cells treated with recombinant CXCL5 (0, 20, and 40 ng/ml) for 48 h were collected and evaluated by cell migration assay. (D) MDA-MB-231 cells treated with recombinant CXCL5 (0, 20, and 40 ng/ml) for 48 h were collected and evaluated by cell invasion assay. (E) MDA-MB-231 cells treated with recombinant CXCL5 (0, 20, and 40 ng/ml) for 48 h were collected and analyzed for the protein expression of mesenchymal marker Slug and cancer stemness marker Oct4 by Western blot. The original blot images in (A) and (E) were available in Supplementary Information. (F,G) CXCL5 neutralizing antibody was added ( +) or omitted (–) during the co-culture of R-ADSCs (R100) or control ADSCs (R0) with MDA-MB-231 cells. After the co-culture, MDA-MB-231 cells were collected and evaluated by cell migration assay in (F) and cell invasion assay in (G). Data were obtained from three independent experiments and presented as mean ± SEM. Statistical difference was determined by t-test comparing R100 group versus their corresponding R0 group as control, or comparing recombinant CXCL5 treatment group (20 and 40 ng/ml) versus recombinant CXCL5 control group (0 ng/ml). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The primary antibodies used in this study for IHC are listed as follows: rabbit polyclonal antibody against human Slug (GeneTex, Hsinchu, Taiwan); goat polyclonal antibody against human CXCL5 (R&D Systems, Minneapolis, MN, USA); mouse monoclonal antibody against human resistin (clone C-10; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibody against human phosphorylated ERK1/2 at Thr202/Tyr204 (clone D13.14.4E; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Co-Culture Assay, Isolation, Control, Enzyme-linked Immunosorbent Assay, Recombinant, Cell Migration Assay, Invasion Assay, Expressing, Marker, Western Blot

Journal: Scientific reports

Article Title: ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

doi: 10.1038/s41598-022-19290-6

Figure Lengend Snippet: Figure 5. Enhanced breast tumor growth and protein expression of CXCL5, Slug, and ERK phosphorylation in mice via xenograft of breast cancer cells after co-culture with resistin-stimulated ADSCs. The isolated ADSCs were treated with resistin at 0 and 50 ng/ml (denoted as R0 and R50, respectively) for 48 h, followed by co-culture with MDA-MB-231 cells in the transwell model for another 72 h. After the co-culture, MDA-MB-231 cells were collected and injected into the fourth mammary fat pads of female NOD/SCID mice for the following analyses. (A) The tumor volume, calculated by (width2 × length)/2, was measured weekly after palpable tumor mass was formed. (B) Upon sacrifice of the mice on week eight, the weight of individual tumor mass was measured. (C) The body weight of the mice was measured weekly. (D) The tumor mass was collected after sacrifice of the mice on week eight, and analyzed for CXCL5, Slug, and phospho-ERK1/2 protein expression by immunohistochemistry (IHC). The quantitative IHC scores were manually evaluated and calculated by multiplying the categorized percentage of stained cells (0, 0–24%; 1, 25‑49%; 2, 50‑74%; 3, 75‑100%) by the categorized intensity of staining (0, negative; 1, weak; 2, moderate; 3, strong). Data were obtained from three to six mice in each group and presented as mean ± SEM or box plots. Statistical difference was determined by t-test comparing R50 group versus R0 group as control. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The primary antibodies used in this study for IHC are listed as follows: rabbit polyclonal antibody against human Slug (GeneTex, Hsinchu, Taiwan); goat polyclonal antibody against human CXCL5 (R&D Systems, Minneapolis, MN, USA); mouse monoclonal antibody against human resistin (clone C-10; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibody against human phosphorylated ERK1/2 at Thr202/Tyr204 (clone D13.14.4E; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Co-Culture Assay, Isolation, Injection, Immunohistochemistry, Staining, Control

Journal: Scientific reports

Article Title: ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

doi: 10.1038/s41598-022-19290-6

Figure Lengend Snippet: Figure 6. Correlation analysis for the protein expression of resistin, CXCL5, and ERK phosphorylation in the tumor and serum specimens from breast cancer patients. (A) Representative images of immunohistochemistry (IHC) staining for resistin, CXCL5, and phospho-ERK1/2 expression in breast tumor sections from breast cancer patients. (B–D) The quantitative IHC scores were evaluated by HistoQuest software, followed by Pearson correlation (r) analysis between resistin and CXCL5 in (B) (n = 96), resistin and phospho-ERK1/2 in (C) (n = 45), and CXCL5 and phospho-ERK1/2 in (D) (n = 45). (E) The serum levels of resistin and CXCL5 in breast cancer patients were determined by ELISA, followed by Pearson correlation (r) analysis (n = 120). The mean ± SD of resistin and CXCL5 was 31.4 ± 15.4 ng/ml and 707.9 ± 293.4 pg/ml, respectively. (F) Schematic summary of the current study. Our preclinical and clinical data together suggest that CXCL5 may be secreted by resistin-stimulated ADSCs in the breast tumor microenvironment, promoting breast cancer cell malignancy via the participation of ERK pathway and epithelial-to-mesenchymal transition. The schematic summary in (F) was produced using the illustration elements from Servier Medical Art (https://smart.servier.com), which is in compliance with the terms of the Creative Commons Attribution 3.0 Unported License (https://creativeco mmons.org/licenses/by/3.0/).

Article Snippet: The primary antibodies used in this study for IHC are listed as follows: rabbit polyclonal antibody against human Slug (GeneTex, Hsinchu, Taiwan); goat polyclonal antibody against human CXCL5 (R&D Systems, Minneapolis, MN, USA); mouse monoclonal antibody against human resistin (clone C-10; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibody against human phosphorylated ERK1/2 at Thr202/Tyr204 (clone D13.14.4E; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Immunohistochemistry, Software, Enzyme-linked Immunosorbent Assay, Produced

Journal: Journal of Translational Medicine

Article Title: CCL20/CCR6 expression profile in pancreatic cancer

doi: 10.1186/1479-5876-8-45

Figure Lengend Snippet: CCL20/CCR6 mRNA expression in pancreatic diseases . Gene expression of [A] CCL20 and [B] CCR6 in chronic pancreatitis (CP, n = 22), pancreatic cystadenomas (PA, n = 11), pancreatic carcinoma (PCA, n = 25) compared to matched normal pancreatic tissues as determined by Q-RT-PCR. Q-RT-PCR data are expressed as mean +/- SEM, *P < 0.05. Fold increase above 1 indicates CCL20/CCR6 up-regulation compared to normal tissues.

Article Snippet: Overnight incubation at 4°C with primary goat polyclonal anti-human CCL20 antibody (15 μg/ml, AF254, R&D Systems, Minneapolis, Minn., USA) was followed by incubation of secondary biotinylated rabbit anti-goat IgG antibody and the avidin-biotin-peroxidase reaction (Vectastain ABC ELITE Kit, Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: CCL20/CCR6 expression profile in pancreatic cancer

doi: 10.1186/1479-5876-8-45

Figure Lengend Snippet: CCL20/CCR6 protein expression in pancreatic diseases . [A] CCL20 protein concentrations (pg/ml pro mg total protein) in chronic pancreatitis (CP, n = 22), pancreatic cystadenomas (PA, n = 11), pancreatic carcinoma (PCA, n = 25) compared to the matched normal pancreatic tissue levels (mean ± SEM), * p < 0.05. [B] Expression of chemokine receptor CCR6 in CP, PA and PCA as determined by Western blot analysis. Total cell lysates of tumor tissues of representative patients of each disease entity were immunoblotted with antibodies specifically recognizing chemokine receptor CCR6. Acute leukemia cell line HL60 served as a positive control for the detection of CCR6. Quantification has been performed using image J software * p < 0.05.

Article Snippet: Overnight incubation at 4°C with primary goat polyclonal anti-human CCL20 antibody (15 μg/ml, AF254, R&D Systems, Minneapolis, Minn., USA) was followed by incubation of secondary biotinylated rabbit anti-goat IgG antibody and the avidin-biotin-peroxidase reaction (Vectastain ABC ELITE Kit, Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Western Blot, Positive Control, Software

Journal: Journal of Translational Medicine

Article Title: CCL20/CCR6 expression profile in pancreatic cancer

doi: 10.1186/1479-5876-8-45

Figure Lengend Snippet: Results of anti-CCL20 immunohistochemistry in normal and diseased pancreatic tissues . Representative example of CCL20 expression in [A,B] pancreatic islet cells and epithelial cells of pancreatic ducts, [C] necrotic parenchyma and epithelial cells of pancreatic ducts in chronic pancreatitis tissues [D] epithelial cells of the characteristic net-like structures of pancreatic cystadenoma [E,F] cytoplasms of ductal epithelial cancer cells and in infiltrates of perineural sheaths. Anti-CCL20 goat anti-human, 75 μg/ml (MIP-3α; R&D Systems; Minneapolis, MN, USA. Avidin Biotin Complex (ABC) Method. (original magnification: × 200 and 400, respectively).

Article Snippet: Overnight incubation at 4°C with primary goat polyclonal anti-human CCL20 antibody (15 μg/ml, AF254, R&D Systems, Minneapolis, Minn., USA) was followed by incubation of secondary biotinylated rabbit anti-goat IgG antibody and the avidin-biotin-peroxidase reaction (Vectastain ABC ELITE Kit, Vector Laboratories, Burlingame, CA, USA).

Techniques: Immunohistochemistry, Expressing, Avidin-Biotin Assay

Journal: Journal of Translational Medicine

Article Title: CCL20/CCR6 expression profile in pancreatic cancer

doi: 10.1186/1479-5876-8-45

Figure Lengend Snippet: Expression of CCL20 in different tumor categories of PCA as determined by [A] Q-RT-PCR and [B] ELISA . mRNA and protein expression profiles of CCL20 (pg/ml pro mg total protein) were measured in PCA tissues and matched normal tissues in pT1 + pT2 (n = 11) (A) and pT3 + pT4 (n = 14) (B), respectively. For Q-RT-PCR data fold increase above 1 indicates CCL20 up-regulation in PCA compared to normal tissues. All data are expressed as mean ± SEM, * p < 0.05

Article Snippet: Overnight incubation at 4°C with primary goat polyclonal anti-human CCL20 antibody (15 μg/ml, AF254, R&D Systems, Minneapolis, Minn., USA) was followed by incubation of secondary biotinylated rabbit anti-goat IgG antibody and the avidin-biotin-peroxidase reaction (Vectastain ABC ELITE Kit, Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay