Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A) HAECs were incubated with the indicated concentrations of CD40L for 24 h, and static adhesion assays were performed as detailed in Methods. Attached THP-1 cells were visualized and counted on an inverted fluorescent microscopy. Magnification, ×20. (B) Quantification of fluorescence density expressed as means ± SEM. * P< 0.05 vs 0 ng/mL. (C) HAECs were incubated in the presence of (40 ng/mL) for the indicated hours, and then static adhesion assays were performed. * P <0.05 vs 0 h. (D) HAECs were incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed with the use of human peripheral monocytes. * P <0.05 vs control. (D) Platelets were activated as described and incubated with HAECs, then THP-1 cells adhesion was analyzed by static adhesion assays. * P <0.05 vs resting platelets.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Microscopy, Fluorescence, Control

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A and B) HAECs were incubated in the presence of indicated concentrations of for 24 hours. A, Total RNA was isolated and subjected to quantitative RT-PCR to analyze VCAM-1 mRNA levels. * P< 0.05 or ** P< 0.01 vs 0 ng/mL. (B) VCAM-1 protein expression was determined by Western blot. Total cell lysates were subjected to SDS-PAGE and immunoblotting. Blots represent 4 independent experiments with similar results. (D) HAECs were pretreated with antibodies (50 μg/mL) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed. * P <0.05 vs CD40L. Data are representative of 4 independent experiments with similar results.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Isolation, Quantitative RT-PCR, Expressing, Western Blot, SDS Page, Control

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A and B) HAECs were incubated in the presence of CD40L (80 ng/mL) or PBS (control) for 24 h, and flow adhesion assays were performed at 37°C. In some experiments, HAECs were pretreated with anti–VCAM-1 antibody (50 μg/mL) for 30 minutes before assay. * P <0.05 vs CD40L. Photographs captured from microscope represent 3 independent experiments with similar results. ×20. (B) Quantification of attached THP-1 cells. * P <0.05 vs CD40L. Data are representative of 4 independent experiments with similar results.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Control, Microscopy

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: HAECs were transfected with vectors encoding the CD40 siRNA or control siRNA and then incubated with CD40L (80 ng/mL) for 24 h. (A) Representative blot showing CD40 protein expression in HAECs transfected with control or CD40 siRNA. (B) VCAM-1 protein expression and (C) THP-1 adhesion were determined as indicated. Data are shown as representative blots or are expressed as the means ± SEM by three independent assays from 4 independent experiments. * P <0.05 vs CD40L plus Control siRNA.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Transfection, Control, Incubation, Expressing

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A) HAECs were transfected with NF-κB reporter for 24 h and then incubated with the indicated concentrations of CD40L for another 8 h. The luciferase activity was determined using β-gal as the control. Results of three independent experiments are expressed as fold of control. * P <0.05, ** P <0.01 vs 0 ng/mL. (B and C) HAECs were stimulated with the indicated concentrations of CD40L for 2 h. Cells were lysed and the protein extracts were assayed for p65 DNA-binding activity. (C) IκB-α protein expression was measured by immunoblotting. (D) HAECs were infected with Ad-IκB, or Ad-GFP for 24 h and then incubated with CD40L (80 ng/ml) for another 24 h. VCAM-1 protein expression was determined by Western blot. The results were reproducible in 4 independent experiments. * P <0.05.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Transfection, Incubation, Luciferase, Activity Assay, Control, Binding Assay, Expressing, Western Blot, Infection

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 2 h. Cytosol and nuclear fractions were prepared to measure NF-κB p65 and IκB-α protein expression by immunoblotting. (B) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h. Total cell lysates were prepared and subjected to immunoblotting. Blots represent 4 independent experiments with similar results.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Control, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

doi: 10.1038/s41598-019-53230-1

Figure Lengend Snippet: ECM niche modulates differential morphology of seeded hAEC. ( A – D ) Representative images of eGFP-hAEC seeded on fibrous or serous side of 6 mm AR-BP discs and imaged after 2 ( A , C ) and 4 ( B , D ) days show that higher number of cells can be observed on serous side compare to fibrous. ( E – H ) When seeded at higher densities and cultured for 2 days, eGFP-hAEC (( E ) 71 cells/mm 2 ; ( F ) 142 cells/mm 2 ; ( G ) 284 cells/mm 2 , ( H ) 568 cells/mm 2 ) seeded on the serous side show multiple vacuoles (arrows) and rounded morphology compared to fibrous side. ( I ) Quantification of cell aspect ratio demonstrates that cells seeded on serous side are statistically more rounded (lower aspect ratio) than those seeded on the fibrous side, and that cell morphology becomes increasingly more rounded with higher seeding densities regardless of seeding side. Scale bar 1 mm ( A , B ), 200 μm ( C – H ). Data represent the mean values natural log-transformed aspect ratios ± SD, n = 5 per group per seeding density (****p < 0.0001).

Article Snippet: Enhanced green fluorescent protein (eGFP) transfected hAEC were purchased from Angio-Proteomie (Boston, MA) (CAT No. cAP‐0006GFP, LOT No. 201209701SR).

Techniques: Cell Culture, Transformation Assay

Journal: Scientific Reports

Article Title: Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

doi: 10.1038/s41598-019-53230-1

Figure Lengend Snippet: Actin expression in eGFP-hAEC seeded on AR-BP scaffolds. Representative microscopic images (n = 5 per group) of eGFP-hAEC (green, first column) after 4 days proliferation and staining for Actin (Phaliodin-594; red, second column) and nuclei (Hoechst 33342; blue, third column). ( A , B ) 71 cell/mm 2 ; ( C , D ) 142 cell/mm 2 ; ( E , F ) 284 cell/mm 2 and ( G , H ) 568 cell/mm 2 . Scale bars: 100 μm (first to fourth column) and 20 μm (fifth column). ( I ) Quantification of cell number on the serous and the fibrous side 4 days after seeding at different densities demonstrate higher cell numbers in the serous side for low and moderate seeding densities (71, 142, and 284 cells/mm 2 ), while cell number equalize when seeded at very high densities (568 cells/mm 2 ). Data represent the average number of cells in a 1.6 mm 2 area ± SD, n = 5 images per group (seeding density and side) (**p < 0.01, *p < 0.05, n.s. non-significant p = 0.4).

Article Snippet: Enhanced green fluorescent protein (eGFP) transfected hAEC were purchased from Angio-Proteomie (Boston, MA) (CAT No. cAP‐0006GFP, LOT No. 201209701SR).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

doi: 10.1038/s41598-019-53230-1

Figure Lengend Snippet: ECM niche modulates hAEC human laminin production following seeding on AR-BP scaffolds. Representative fluorescent microscopy images of ( A’ – D’ ) cell nuclei (blue), ( A” – D” ) eGFP-hAEC (green), and ( A”’ – D”’ ) human laminin (red). The serous side was found to have a continuous laminin layer while on the fibrous side new laminin production was found to be strongly associated with areas of cellular adhesion, especially at low seeding densities (n = 5 per group). ( E , F ) Quantification of human laminin production per cell. At moderate seeding density (142 cells/mm 2 ) hAEC seeded on the serous side produce more laminin per cell than those seeded on the fibrous side (p = 0.0002). At high seeding density (568 cells/mm 2 ) the effect of ECM niche on per cell laminin production is no longer present. Scale bar = 10 µm.

Article Snippet: Enhanced green fluorescent protein (eGFP) transfected hAEC were purchased from Angio-Proteomie (Boston, MA) (CAT No. cAP‐0006GFP, LOT No. 201209701SR).

Techniques: Microscopy