Journal: Oncotarget

Article Title: ΔNp63 isoform-mediated β-defensin family up-regulation is associated with (lymph)angiogenesis and poor prognosis in patients with squamous cell carcinoma.

doi: 10.18632/oncotarget.1819

Figure Lengend Snippet: Figure 5: ΔNp63-regulated HβDs enhance endothelial cell migration. A: CD105 immunostaining showing the neoformed blood vessel density in high and low ΔNp63-expressing SCC. Increased counts of CD105+ blood vessels were detected in high ΔNp63- expressing cervical and head and neck SCC when compared to tumors with a weakly positive ΔNp63 immunoreactivity (B). The average number of blood vessels per mm2 was measured as described in the “Materials and Methods” section. Influence of HβDs on endothelial cell migration in a Boyden Chamber assay (C), viability (E) and proliferation (F). The HβDs-related migratory ability of blood vessel cells was also measured with a CCR6-blocking antibody (D). PBS and VEGF-A were used as negative and positive control, respectively. Data are presented as the means ± standard deviation of four different experiments. G: Enhancement of angiogenesis by HβDs in the CAM assay. Representative images of alpha-SMA positive-blood vessels are shown. Quantification of blood vessels has been performed by computerized image analysis. (H) Relative vascular area and (I) number of blood vessels per mm2 were determined. Asterisks indicate statistically significant differences (*P < 0.05).

Article Snippet: For inhibitory experiments, blocking anti-CCR6 antibody (Clone 53103, R and D systems) was added 20 min at 37 °C before treatments were started.

Techniques: Migration, Immunostaining, Expressing, Boyden Chamber Assay, Blocking Assay, Positive Control, Standard Deviation, Chick Chorioallantoic Membrane Assay

Journal: Oncotarget

Article Title: ΔNp63 isoform-mediated β-defensin family up-regulation is associated with (lymph)angiogenesis and poor prognosis in patients with squamous cell carcinoma.

doi: 10.18632/oncotarget.1819

Figure Lengend Snippet: Figure6:ΔNp63-regulated HβDs promote lymphangiogenesis. A: Anti-Podoplanin antibody was used to determine the lymphatic vessel density in high and low ΔNp63-expressing SCC. Increased counts of podoplanin+ lymphatic vessels were observed in high ΔNp63- expressing cervical and head and neck SCC when compared to neoplastic lesions with a weakly positive ΔNp63 immunoreactivity (B). The average number of lymphatic vessels per mm2 was determined. Influence of HβDs on lymphatic endothelial cell migration (C), viability (E) and proliferation (F) was analyzed as described in the Materials and Methods section. Boyden chamber assay of lymphatic endothelial cells with CCR6-blocking antibody was also performed (D). PBS and VEGF-C were used as negative and positive control, respectively. Data are presented as the means ± standard deviation of four different experiments. G: Enhancement of lymphangiogenesis by HβDs in the CAM assay. Representative images of Prox1 positive-blood vessels are shown. Quantification of lymphatic vessels has been performed by computerized image analysis. (H) Relative vascular area and (I) number of lymphatic vessels per mm2 were determined. Asterisks indicate statistically significant differences (*P < 0.05).

Article Snippet: For inhibitory experiments, blocking anti-CCR6 antibody (Clone 53103, R and D systems) was added 20 min at 37 °C before treatments were started.

Techniques: Expressing, Migration, Boyden Chamber Assay, Blocking Assay, Positive Control, Standard Deviation, Chick Chorioallantoic Membrane Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: CyTOF antibody panel

Article Snippet: 141Pr , CD196 (CCR6) , 11A9 , Fluidigm.

Techniques:

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: 141Pr , CD196 (CCR6) , 11A9 , Fluidigm.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software

Journal: Journal of Functional Foods

Article Title: Membrane vesicles from the probiotic Nissle 1917 and gut resident Escherichia coli strains distinctly modulate human dendritic cells and subsequent T cell responses

doi: 10.1016/j.jff.2019.103495

Figure Lengend Snippet: Fig. 6. Quantification of the Th17 and Treg subsets induced by MV-stimulated DCs. (A-B) Flow cytometric analysis of CD4+ T cells co-incubated for 3 days with untreated mo-DCs (iDC) or mo-DCs primed with MVs of the indicated strains (10 μg/ml) at a DC:T cell ratio of 1:5 with specific markers for: (A) Th17 population (CD4+ CD196+) and (B) Treg (CD25+(High) FoxP3+) population. Representative dot plots of CD4+ cells co-incubated with iDCs (control) or with EcN MV- stimulated DCS are shown for each cell subset analysis. The percentage of positive cells is indicated in each quadrant or box. (C) Data (mean ± SEM) from three- independent experiments performed in triplicate are expressed as fold-change increase in the percentage of positive T cells for Th17 or Treg specific markers induced by MV-stimulated DCs with respect control iDCs induced values (set as 1, dot line). Statistical differences were evaluated by one-way ANOVA followed by Bonferroni’s test (normal distribution) *p < 0.05 versus control DCs.

Article Snippet: For surface marker staining, lymphocytes were first stained with anti-CD4 FITC (clone VIT4), anti-CD25 APC (clone 4E3) and anti-CD196 PE (clone REA190) mAb or with the corresponding isotype-matched conjugated irrelevant antibody (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Incubation, Control

Journal: Cell

Article Title: NKG2A blockade potentiates CD8 T-cell immunity induced by cancer vaccines

doi: 10.1016/j.cell.2018.10.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CCR6 – 141 Pr (G034E3) , Fluidigm , Prod#: 3141003A; RRID: AB_2687639.

Techniques: Blocking Assay, Purification, Virus, Recombinant, Staining, Transfection, Cell Stimulation, Adjuvant, Mutagenesis, Transgenic Assay, Marker, Sequencing, Expressing, Plasmid Preparation, Software, CRISPR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL20 and β-defensin-2 induce arrest of human Th17 cells on inflamed endothelium in vitro under flow conditions.

doi: 10.4049/jimmunol.1000597

Figure Lengend Snippet: FIGURE 4. Specific ligand-mediated triggering of CCR6 induces CD11a/CD18-CD54–dependent arrest of differentiated Th17 cells on HUVEC, stimulated with IL-1b and TNF-a, in vitro under flow conditions. Fully differentiated human Th17 and Th2 clones (A–C) or purified CCR6+ and CCR62

Article Snippet: mAbs, flow cytometry analysis, and FACS sorting The following mAbs were used for immunostaining: FITC- or PE-conjugated anti-CD4 mAbs (BD Biosciences, Le Pont-De-Claix, France), a FITC- or PE-conjugated anti-CCR6 mAb (R&D Systems, Lille, France), and FITC-conjugated anti-CD54, CD62E, and CD106 mAbs (BD Biosciences).

Techniques: In Vitro, Clone Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL20 and β-defensin-2 induce arrest of human Th17 cells on inflamed endothelium in vitro under flow conditions.

doi: 10.4049/jimmunol.1000597

Figure Lengend Snippet: FIGURE 5. Calcium mobilization in CCR6-expressing CHO cells by CCL20 and hBD-2. CHO cells stably transfected with human CCR6 re- ceptor were loaded with Ca No Wash Plus Dye and stimulated with CCL20 and hBD-2. To study CCR6 receptor desensitization, 350 ng/ml CCL20 was added 60 s after the first stimulation. [Ca2+]i concentration was monitored by measuring fluorescence for 180 s on a fluorescence plate reader. A, Ki- netics study obtained with CCL20 and hBD-2 at 250 and 500 ng/ml final concentration, respectively. Arrowheads indicate time of application of each ligand. [Ca2+]i response was calculated as relative fluorescence units (RFU) minus background from each value. B, The inhibitory activity of hBD-2 (filled squares) and CCL20 (empty squares) was assayed at indicated con- centrations on calcium response elicited in CHO-CCR6 by 350 ng/ml CCL20. Data are representative of three independent experiments.

Article Snippet: mAbs, flow cytometry analysis, and FACS sorting The following mAbs were used for immunostaining: FITC- or PE-conjugated anti-CD4 mAbs (BD Biosciences, Le Pont-De-Claix, France), a FITC- or PE-conjugated anti-CCR6 mAb (R&D Systems, Lille, France), and FITC-conjugated anti-CD54, CD62E, and CD106 mAbs (BD Biosciences).

Techniques: Expressing, Stable Transfection, Transfection, Concentration Assay, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL20 and β-defensin-2 induce arrest of human Th17 cells on inflamed endothelium in vitro under flow conditions.

doi: 10.4049/jimmunol.1000597

Figure Lengend Snippet: FIGURE 6. Ag-specific activation results in downregulation of CCR6 mRNA and loss of CCR6 expression from the surface of Th17 cells. A Th17 cell clone was stimulated with the combination of anti-CD3 and anti-CD28 mAbs (A), or a PPD-specific CCR6-expressing T cell clone was stimulated with PPD and autologous monocytes (B), and the evolution of CCR6 expression at the cell surface was analyzed by flow cytometry. The x-axis represents fluorescence (four-decade log scale), and the y-axis represents the relative cell number. Values in histograms represent percentage of CCR6+ T cells and mean fluo- rescence intensity. Isotype-matched control mAb as in Fig. 4D. C, Cells, stimulated as under A, were collected at the indicated time interval after their activation, lysed, and CCR6 mRNA expression was determined by real-time PCR. Values represent mean 6 SD of three experiments.

Article Snippet: mAbs, flow cytometry analysis, and FACS sorting The following mAbs were used for immunostaining: FITC- or PE-conjugated anti-CD4 mAbs (BD Biosciences, Le Pont-De-Claix, France), a FITC- or PE-conjugated anti-CCR6 mAb (R&D Systems, Lille, France), and FITC-conjugated anti-CD54, CD62E, and CD106 mAbs (BD Biosciences).

Techniques: Activation Assay, Expressing, Cytometry, Control, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL20 and β-defensin-2 induce arrest of human Th17 cells on inflamed endothelium in vitro under flow conditions.

doi: 10.4049/jimmunol.1000597

Figure Lengend Snippet: FIGURE 7. Downregulation of CCR6 expression on Th17 cells is independent from endogenous CCL20 production. Th17 cells were incubated in medium only or in the presence of CCL20 (100 ng/ ml) or were activated with anti-CD3 and anti-CD28 mAb. CCR6 expression was analyzed at the indicated time periods by flow cytometry. A, Flow cytometry analysis data from a representative experiment. The x-axis represents fluorescence (four-decade log scale), and the y-axis represents the relative cell number. Values indicated in the upper right quadrants repre- sent the percentage 6 SD of CCR6-positive cells and their relative fluorescence intensity. B, Evolution of cell-surface CCR6 expression expressed as the rel- ative fluorescence intensity 6 SD obtained in three independent experiments.

Article Snippet: mAbs, flow cytometry analysis, and FACS sorting The following mAbs were used for immunostaining: FITC- or PE-conjugated anti-CD4 mAbs (BD Biosciences, Le Pont-De-Claix, France), a FITC- or PE-conjugated anti-CCR6 mAb (R&D Systems, Lille, France), and FITC-conjugated anti-CD54, CD62E, and CD106 mAbs (BD Biosciences).

Techniques: Expressing, Incubation, Cytometry, Flow Cytometry

Journal: OncoTargets and therapy

Article Title: Oncogenic Role and Prognostic Value of MicroRNA-937-3p in Patients with Breast Cancer

doi: 10.2147/OTT.S229510

Figure Lengend Snippet: miR-937-3p negatively regulates CCRL2 expression by directly targeting its 3ʹ UTR. Notes: Venn diagram of target genes for miR-937-3p predicted by targetscan and miRDB website ( A and B ). The predicted binding sites of miR-937-3p on the 3′UTR of CCRL2 and the mutant miR-937-3p sequence at the binding site ( C ). The miR-937-3p expression level in breast cancer tissues and adjacent normal breast tissues were compared using TCGA database ( D ) and GSE70905 database ( E ). Correlations between miR-937-3p and CCRL2 mRNA levels ( F ). The analysis of the relative luciferase activity of CCRL2-3′UTR plus miR-937-3p, CCRL2-3′UTR plus miR-937-3p Mut and CCRL2-3′UTR plus miR-937-3p-NC ( G ). Relative expression of CCRL2 protein in MCF-7 cells transfected with miR-937-3p mimics, miR-937-3p Mut or miR-937-3p-NC ( H and I ). **p<0.01; ***p<0.001. Abbreviation: NS, not significant.

Article Snippet: Anti-GAPDH antibody (60004-1-Ig) and anti-CCRL2 antibody (66611-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Activity Assay, Transfection

Journal: OncoTargets and therapy

Article Title: Oncogenic Role and Prognostic Value of MicroRNA-937-3p in Patients with Breast Cancer

doi: 10.2147/OTT.S229510

Figure Lengend Snippet: miR-937-3p promotes cell proliferation, migration and invasion through regulating CCRL2 in breast cancer. Notes: The expression of CCRL2 in MCF-7 cells transfected with CCRL2-siRNA or CCRL2-siRNA-NC was determined by RT-qPCR ( A ) and western-blot ( B ). CCK8 and SRB assay were used to evaluate the proliferation of MCF-7 cells following transfection with miR-937-3p-inhibitor plus CCRL2-siRNA or CCRL2-siRNA-NC transfection ( C and D ). The migration ability of MCF-7 cells with miR-937-3p-inhibitor plus CCRL2-siRNA or CCRL2-siRNA-NC transfection ( E and F ). The invasion ability of MCF-7 cells with miR-937-3p-inhibitor plus CCRL2-siRNA or CCRL2-siRNA-NC transfection ( G and H ). **p<0.01; ***p<0.001.

Article Snippet: Anti-GAPDH antibody (60004-1-Ig) and anti-CCRL2 antibody (66611-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Migration, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Sulforhodamine B Assay

Journal: OncoTargets and therapy

Article Title: Oncogenic Role and Prognostic Value of MicroRNA-937-3p in Patients with Breast Cancer

doi: 10.2147/OTT.S229510

Figure Lengend Snippet: miR-937-3p is an unfavorable prognostic factor in breast cancer. Notes: The Kaplan–Meier survival analyses of miR-937-3p expression of the overall survival time in breast cancer patients using TCGA ( A ). The Kaplan–Meier survival analyses of CCRL2 expression of the overall survival time in breast cancer patients using GSE16446 based on the website of the Kaplan–Meier plotter ( B ).The Kaplan–Meier survival analyses of miR-937-3p low level and CCRL2 high level, miR-937-3p high level and CCRL2 low level and other (miR-937-3p low level and CCRL2 low level and miR-937-3p high level and CCRL2 high level) in breast cancer patients using TCGA ( C ).

Article Snippet: Anti-GAPDH antibody (60004-1-Ig) and anti-CCRL2 antibody (66611-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing