Journal: Biomolecules

Article Title: CD69 Signaling in Eosinophils Induces IL-10 Production and Apoptosis via the Erk1/2 and JNK Pathways, Respectively

doi: 10.3390/biom14030360

Figure Lengend Snippet: CD69 expression and cytokines profiling of mouse eosinophils. ( A ) Surface expression of isotype IgG and CD69 on peripheral blood and lung eosinophils in the healthy and asthmatic mouse models using flow cytometry ( n = 6). ( B ) Immunofluorescence staining of healthy and asthmatic lung tissue sections stained with antibodies, with corresponding isotypes IgG, Siglec-F, and CD69. ( C ) Surface CD69 expression on eosinophils from peripheral blood and the spleen in naive IL-5 transgenic (Tg) mice ( n = 5 for each group). ( D ) CD69 expression observed using immunofluorescence staining in the eosinophils purified from the blood and spleen of naive IL-5Tg mice. ( E ) qPCR analysis of Th1, Th2, and Th17 cytokines; transcription factors; IL-10; and TGF-β mRNA expression in the CD69 + eosinophils purified using a cell sorter. Relative gene expressions were calculated with ΔΔCt method using sorted CD69 − eosinophils as the reference ( n = 4 for each group). Data are expressed as mean ± SEM. * indicates a significant difference compared with healthy mice, blood, or CD69 − eosinophils ( p < 0.05).

Article Snippet: Briefly, purified eosinophils were incubated with 10 ng, 100 ng, 1 μg, or 10 μg/mL of anti-mouse CD69 mAb (Novus Biologicals, Centennial, CO, USA) or with Armenian hamster IgG (BioLegend, San Diego, CA, USA) as the control at 4 °C for 30 min. Then, cells were transferred into a 24-well plate coated with 20 μg/mL of goat anti-Armenian hamster IgG as a secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Staining, Transgenic Assay, Purification

Journal: Biomolecules

Article Title: CD69 Signaling in Eosinophils Induces IL-10 Production and Apoptosis via the Erk1/2 and JNK Pathways, Respectively

doi: 10.3390/biom14030360

Figure Lengend Snippet: Functional role and signaling pathway of CD69 on eosinophils following CD69 crosslinking. ( A ) qPCR analysis of the Th1, Th2, and Th17 cytokines, transcription factors, IL-10, and TGF-β mRNA expression at 1, 3, and 6 h after 10 μg/mL CD69 crosslinking on eosinophils. The mRNA expressions on CD69 + eosinophils were compared with those on non-stimulated eosinophils (isotype), which were considered as reference ( n = 3 for each group). ( B , C ) mRNA expression and protein level of IL-10 after corresponding isotype IgG, 10 ng, 100 ng, 1 μg, or 10 μg/mL CD69 crosslinking on eosinophils for 3 h and 24 h, respectively ( n = 4 for each group). * indicates a significant difference from isotype group ( p < 0.05). ( D ) Phosphorylation and total Erk1/2, STAT5, Jak3, and JNK protein and β-actin expression in eosinophils purified from IL-5 Tg mouse following crosslinking by 10 μg/mL CD69 antibody or matched control via Western blot analysis. IL-10 secretion ( E ) and % survival of eosinophils ( F ) following 10 μg/mL CD69 crosslinking on eosinophils purified from the spleen of IL-5 Tg mice for 24 h culture in the absence (positive control group) or presence of 50 μM PD98059 ( n = 4) and 50 µM SP600125 ( n = 4), respectively. Data are expressed as mean ± SEM. * indicates a significant difference from 10 μg/mL anti-CD69 stimulation without inhibitor treatment group (positive control group) using a two-way ANOVA ( p < 0.05). Original figures can be found in .

Article Snippet: Briefly, purified eosinophils were incubated with 10 ng, 100 ng, 1 μg, or 10 μg/mL of anti-mouse CD69 mAb (Novus Biologicals, Centennial, CO, USA) or with Armenian hamster IgG (BioLegend, San Diego, CA, USA) as the control at 4 °C for 30 min. Then, cells were transferred into a 24-well plate coated with 20 μg/mL of goat anti-Armenian hamster IgG as a secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Functional Assay, Expressing, Phospho-proteomics, Purification, Control, Western Blot, Positive Control