Journal:

Article Title: Accelerated chemokine receptor 7-mediated dendritic cell migration in Runx3 knockout mice and the spontaneous development of asthma-like disease

doi: 10.1073/pnas.0504787102

Figure Lengend Snippet: Dysregulated expression of CCR7 in Runx3 KO DC. (A) Impaired TGFβ-dependent inhibition of CCR7 transcription in KO BMDC. WT and Runx3 KO BMDC were grown in the presence or absence of TGFβ (10 ng/ml). At day 6, cells were induced to undergo maturation by LPS, and, at day 7, RNA was prepared and analyzed by RT-PCR. (B) Impaired TGFβ-dependent inhibition of surface expression of CCR7 in KO BMDC. WT and Runx3 KO BMDC were grown and treated as in A. At day 7, cells were analyzed by FACS by using anti-CCR7 antibodies (goat anti-mouse CI0131, Capralogics). FSChighCD11c+ DC were gated, and their CCR7 expression was determined. Reduction in the level of surface CCR7 and in the number of cells expressing it was noted only in WT and not in Runx3 KO BMDC. (C and D) Increased CCR7 expression on alveolar and LN DC of Runx3 KO mice. BAL and peripheral LN cells of KO and WT mice (n = 3) were obtained and analyzed. (C) Alveolar DC (FSChigh/CD11chigh) were gated and analyzed for CCR7 expression. Of note, expression of CCR7 on the DC subpopulation CD11c+/CD11b+ present only in KO lungs is shown along with that of KO CD11c+/CD11b- DC. (D) FSChigh/CD11chigh DC of axillary and thoracic LN were gated and analyzed for CCR7 expression.

Article Snippet: At day 7, cells were analyzed by FACS by using anti-CCR7 antibodies (goat anti-mouse CI0131, Capralogics).

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Accelerated chemokine receptor 7-mediated dendritic cell migration in Runx3 knockout mice and the spontaneous development of asthma-like disease

doi: 10.1073/pnas.0504787102

Figure Lengend Snippet: Elevated CCR7 mediated in vivo trafficking of alveolar DC to the draining LN in the KO mice. (A and B) Runx3 KO (n = 7) and WT (n = 5) mice were treated by intranasal administration of CFSE to label in vivo the respiratory DC. When indicated, WT mice were treated with LPS (n = 4), and Runx3 KO mice were treated with anti-CCR7 antibody (n = 4) or with buffer only (n = 4). KO mice (n = 4) and LPS-treated WT mice (n = 3) were also treated by inhalation of Ciglitazone. Eighteen hours later, mice were killed, and single-cell suspensions of BAL, thoracic LN, and axillary LN were prepared and analyzed by FACS. (A) FSChigh/CD11c+ DC were gated (R1 and R2). Shown is representative side scatter (SSC) versus CFSE staining of DC populations in BAL and LN after the various treatments. (B) Migration index of alveolar DC to thoracic LN represents the ratio between the percentage of CFSE+ cells within the CD11c+ population in the thoracic LN and the respective value in BAL cells. Results are presented as mean ± SEM. Analysis of variance showed that the migration index of untreated KO DC was significantly higher than that of WT (*, P = 0.016). Notably, the anti-CCR7-treated KO DC migration index was similar to basal migration of WT, and Ciglitazone significantly (*, P = 0.03) reduced the migration of KO DC.

Article Snippet: At day 7, cells were analyzed by FACS by using anti-CCR7 antibodies (goat anti-mouse CI0131, Capralogics).

Techniques: In Vivo, Staining, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: Co-expression of CCR7 and MUC1 in ESCC sample. a The expression of CCR7 and MUC1 in ESCC detected by IHC; b the immunoreactivity score of MUC1 in group with CCR7 positive expression and CCR7 negative expression group; c the 3-year regional recurrence curve of patients with CCR7 and MUC1 positive/negative expression; d the 5-year survival curve of patients with CCR7 and MUC1 positive/negative expression

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: Clinical characteristics and its relationship with CCR7/ MUC1 expression

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: Clinical characteristics and its relationship with CCR7 & MUC1 expression

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: CCL21 induced the up-regulation of MUC1 in ESCC cell lines. a , b the mRNA and protein expression level of MUC1 and CCR7 in ESCC cell lines; c : CCL21 induced the increasing MUC1 mRNA in ESCC cells. KYSE150, KYSE410, KYSE450 and Eca9706 were starved for 24 h and then treated with CCL21 at a concentration of 100 ng/ml for 12 h, then cells were harvested for the qRT-PCR; d , e CCL21 up regulated mRNA of MUC1 in a time and dose dependent way. Eca9706 and KYSE410 were starved for 24 h and then treated with CCL21 at a concentration of 0, 25, 50, 100, 200 ng/ml for 12 h or cells were treated with CCL21 at a concentration of 100 ng/ml for 0, 2, 6, 12 or 24 h. Then cells were harvested for qRT-PCR; f , g increasing of MUC1-C in KYSE410 and Eca9706 after treated with CCL21 at concentration of 0, 25,50, 100, 200 ng/ml for 24 h confirmed by immunoblotting; h , f blocking CCR7suppressed the up-regulation of MUC1 induced by CCL21. Eca9706 and KYSE410 were pretreated by CCR7 antibody (1 ug/mL) or IgG as control, then treated by 100 ng/ml for 24 h, then cells were harvested for detection of MUC1 by immunoblotting; j expression of MUC1 in KYSE410 and Eca9706 treated with PBS or CCL21(100 ng/mL) detected by immunofluorescence. Each data point represents the mean ± SD of three repeated experiments. * P < 0.05

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Western Blot, Blocking Assay, Control, Immunofluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: Silencing of MUC1 suppressed migration and invasion induced by CCL21. Cells were starved for 24 h and then seeding into the upper chamber, for the migration assay CCL21 was added into the lower chamber at a concentration of 200 ng/ml and incubated for 12 h; for the invasion assay the CCL21 was added into the upper chamber at a concentration of 200 ng/ml and incubated for 36 h. Cells on the lower surface of the membrane were counted in five randomly selected fields. a CCL21 promoted migration and invasion of KYSE410 and Eca9706 while blocking CCR7 could reverse migration and invasion induced by CCL21, and silencing MUC1 by siRNA targeted to MUC1 significantly suppressed the migration and invasion induced by CCL21; b Total cell numbers on the lower surface of the membrane counted in five randomly selected fields. Each data point represents the mean ± SD of 3 repeated experiments. * P < 0.05; Each datapoint represents the mean ± SD of three repeated experiments. * P < 0.05

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques: Migration, Concentration Assay, Incubation, Invasion Assay, Membrane, Blocking Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: Activation of ERK1/2 was responsible for the up-regulation of MUC1 induced by CCL21-CCR7. a The activation of ERK1/2 and Akt pathway induced by CCL21, KYSE410 and Eca9706 cells were seeding into 6 well culture plate and starved overnight, then culture medium was replaced by serum free medium contained CCL21 (100 ng/ml) and incubated for 0, 15, 30, 45, 60 min. Then cells were harvested for immunoblotting; b Blocking CCR7 could suppress the activation of Akt and ERK1/2 pathway induced by CCL21 in KYSE410 and Eca9706; c , d Inhibiting activation of ERK1/2 but not Akt could suppress the up-regulation of MUC1-C protein. The starved KYSE410 and Eca9706 cells were pretreated by DMSO as controll, U0126 or MK2206 for 30 min, then cells were treated with PBS or CCL21 (100 ng/ml). For detecting p-ERK1/2, p-Akt and MUC1-C, cells were harvested after incubated with CCL21 for 15 min, 30 min and 24 h respectively; e Inhibiting ERK1/2 but not Akt could remarkably suppress the activity of MUC1 promoter, KYSE410 and Eca9706 cells transfected with MUC1-pGL2b plasmid were pretreated with DMSO as control, U0126 or MK2206 and then treated with PBS or CCL21 (100 ng/ml) for 12 h, then cells were harvested to detect the relative luciferase activity. Each datapoint represents the mean ± SD of three repeated experiments. * P < 0.05

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques: Activation Assay, Incubation, Western Blot, Blocking Assay, Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: Phosphorylation of SP1 was responsible for the up-regulation of MUC1 induced by CCL21-CCR7. a , b Silencing Sp1 could remarkably suppressed the up-regulation of MUC1 in KYSE410 and Eca9706 induced by CCL21. The starved siSp1-Eca9706, siNC-Eca9706, siSp1-KYSE410 and siNC-KYSE410 were treated with CCL21(100 ng/mL) for 24 h, then cells were harvested for immunoblotting for MUC1-C; c Silencing Sp1/Mutant of Sp1 binding site at −99/−97 could remarkably suppressed MUC1 promoter activity induced by CCL21. siSp1-Eca9706, siNC-Eca9706, siSp1-KYSE410 and siNC-KYSE410 transfected by MUC1-pGL2b luciferase reporter plasmid/KYSE410 and Eca9706 cells transfected by MUC1-pGL2b or MUC1 mutant-pGL2b -firefly luciferase reporter plasmid were treated with PBS or CCL21(100 ng/mL) for 24 h, then cells were harvested for detecting the luciferase activity; d Increasing phosphorylation of Sp1 induced by CCL21. Starved KYSE410 and Eca9706 cells were treated with CCL21(100 ng/mL) for 0, 0.5, 1,2 and 6 h, then cells were harvested for the immunoblot of p-Sp1; e The expression of p-Sp1 in KYSE410 treated with PBS or CCL21(100 ng/ml) for 6 h detected by immunofluorescence; f Blocking CCR7 could suppress the phosphorylation of Sp1 induced by CCL21. The starved KYSE410 and Eca9706 cells pretreated with the CCR7 antibody or IgG as control, were treated with PBS or CCL21 for 6 h, then cells were harvested for the immunoblot of p-Sp1; g Inhibiting ERK1/2 suppressed phosphorylation of SP1 induced by CCL21. Starved KYSE410 and Eca9706 cells were pretreated with DMSO as control or U0126 for 30 min. After treated with PBS or CCL21(100 ng/mL) for 6 h, the cells were harvested for immunoblot; h Inhibiting ERK1/2 suppressed Sp1 binding to MUC1 promoter at −99/−97. Starved KYSE410 cells were pretreated with DMSO as control or U0126 for 0.5. After treated with PBS or CCL21(100 ng/mL) for 12 h, the cells were harvested for the ChIP assay. The targeted DNA was amplified using MUC1 primers with 40 cycles of PCR. Each datapoint represents the mean ± SD of three repeated experiments. * P < 0.05

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques: Phospho-proteomics, Western Blot, Mutagenesis, Binding Assay, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Immunofluorescence, Blocking Assay, Control, Amplification

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1

doi: 10.1186/s13046-015-0268-9

Figure Lengend Snippet: Heterologous CCR7 promoted migration and invasion and up-regulated expression of MUC1 in KYSE150. a Heterologous expression of CCR7 up regulated the expression of MUC1 mRNA in KYSE150, KYSE150-CCR7 and KYSE150NC cells were starved and then treated with PBS or CCL21(0, 25, 50, 100, 200 ng/mL) for 12 h and then harvested for qRT-PCR and the result showed the remarkable up-regulation of MUC1 in KYSE150-CCR7 after treated with CCL21 compared to the KYSE150NC groups; b Heterologous expression of CCR7 up regulated the expression of MUC1-C protein in KYSE150, KYSE150-CCR7 and KYSE150NC cells were starved and then treated with PBS or CCL21(100 ng/mL) for 24 h and then harvested for immunoblot and the result showed the remarkable up-regulation of MUC1 in KYSE150-CCR7 after treated with CCL21 compared to the KYSE150NC groups; c Heterologous expression of CCR7 promoted migration and invasion induced by CCL21, the starved KYSE150-CCR7 and KYSE150NC cells were seeding into the upper chamber, for the migration assay CCL21 was added into the lower chamber at a concentration of 200 ng/ml and incubated for 12 h; for the invasion assay the CCL21 was added into the upper chamber and incubated for 36 h; d Total cell numbers on the lower surface of the membrane counted in five randomly selected fields. Each data point represents the mean ± SD of three repeated experiments. * P < 0.05

Article Snippet: Specific mouse polyclonal anti-MUC1 antibody (Abcam, UK) and specific rabbit polyclonal anti-CCR7 antibody (Abcam, UK) were used at a dilution of 1:100 and incubated at 4 °C overnight.

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Incubation, Invasion Assay, Membrane

Journal: Acta biomaterialia

Article Title: Design principles for cytokine-neutralizing gels: Cross-linking effects

doi: 10.1016/j.actbio.2010.06.029

Figure Lengend Snippet: Quantification of immunohistochemical staining of tissue sites treated as noted. Bars shaded with vertical lines were stained for the pan-macrophage marker CD68, bars with diagonal shading were stained for the macrophage M2 phenotype marker CD163 and bars shaded solid black were stained for the macrophage M1 marker CCR7. Select p-values are written above the double-headed arrows connecting results that have significantly different values.

Article Snippet: The secondary antibodies used were biotinylated horse anti-mouse IgG (Vector, CD68 and CD163) at a dilution of 1:50 and goat anti-rabbit IgG (CCR7, Vector) at a dilution of 1:100.

Techniques: Immunohistochemical staining, Staining, Marker

Journal: Molecular Therapy Oncolytics

Article Title: The IAP antagonist birinapant enhances chimeric antigen receptor T cell therapy for glioblastoma by overcoming antigen heterogeneity

doi: 10.1016/j.omto.2022.11.004

Figure Lengend Snippet: Birinapant does not significantly affect CAR T cell functions in vivo NSG mice were subcutaneously implanted with 0.5 × 10 6 birinapant-resistant U87-EGFRvIII-CBG cells on the right flank. After 6 days (day 0), 5 × 10 6 2173-28ζ CAR T cells were intravenously injected. The vehicle (12.5% Captisol) or birinapant at 10 mg/kg were given by intraperitoneal injections every 3 days for a total of 10 doses since day 0. The mice were rechallenged with 3 × 10 6 U87-EGFRvIII-CBG cells subcutaneously implanted in the left flank on day 22. (A) Geometric mean of bioluminescent total flux by imaging from U87-EGFRvIII-CBG tumors in each treatment group at the indicated time points. (B) Concentration of TNF-α and IFN-γ in mouse plasma on day 7 by ELISA. (C) Geometric mean of human CAR T cell (CD45 + /CD3 + ) count in mouse blood in each treatment group at the indicated time points. (D) Percentage of CD4 (CD4 + /CD8 − ), CD8 (CD4 − /CD8 + ), naïve-like (CCR7 + /CD45RO − ), central memory (CCR7 + /CD45RO + ), effector memory (CCR7 − /CD45RO + ), and effector (CCR7 − /CD45RO − ) T cell subset among total human CAR T cells (CD45 + /CD3 + ) in mouse blood on day 21 by flow cytometry. BLI, bioluminescent imaging. p values were determined by unpaired t tests in (B). Results are presented as geometric means ± geometric SD in (A) and (C), and as means ± SD in (B). ∗p < 0.05.

Article Snippet: For flow cytometry with mouse blood samples to evaluate human CAR T cell count and phenotypes in mouse studies, the staining panel included mouse-anti-human CD45-APC (BD Biosciences; clone HI30), mouse-anti-human CD3-BV605 (BioLegend; clone OKT3), mouse-anti-human CD4-BV510 (BioLegend; clone OKT4), mouse-anti-human CD8-APC-H7 (BD Biosciences; clone SK1), mouse-anti-human CCR7-FITC (BD Biosciences; clone 150503), and mouse-anti-human CD45RO-PE (BioLegend; clone UCHL1), and a separate panel was composed of mouse-anti-human PD1-BV421 (BioLegend; clone EH12.2H7), mouse-anti-human LAG3-PerCP/Cyanine5.5 (BioLegend; clone11C3C65), rabbit-anti-human TIM3-AF700 (R&D Systems; clone 344823), and mouse-anti-human CD45-FITC (BD Biosciences; clone HI30).

Techniques: In Vivo, Injection, Imaging, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The BCR-ABL inhibitor nilotinib influences phenotype and function of monocyte-derived human dendritic cells

doi: 10.1007/s00262-018-2129-9

Figure Lengend Snippet: Migratory capacity of DCs to CCR7 ligand. Imatinib- and nilotinib-treated and LPS-stimulated human DCs were analyzed for their migratory behavior towards CCL19/MIP-3β in transwell assays

Article Snippet: Immunostaining Cells were stained using FITC-, BB515 (brilliant blue 515) or PE-conjugated mouse monoclonal antibodies against CD14, CD80, HLA-DR (human leukocyte antigen–antigen D related), PD-1 (BD Biosciences, Heidelberg, Germany), PD-L1 (BioLegend, Koblenz, Germany), CD86, CTLA-4 (BD PharMingen, Hamburg, Germany), CD1a (DAKO Diagnostika GmbH, Hamburg, Germany), CD83 (Immunotech, Marseilles, France), CCR7, RANK (receptor activator of NF-κB, R&D Systems), and CD209 (BD Biosciences).

Techniques: