Journal: International Journal of Molecular Sciences

Article Title: ROCK2 Regulates Monocyte Migration and Cell to Cell Adhesion in Vascular Endothelial Cells

doi: 10.3390/ijms20061331

Figure Lengend Snippet: ROCK modulates expression of inflammatory cytokines, receptors and adhesion molecule. ( A ) Inflammatory cytokines and receptors PCR array for HAECs. No.1–3 samples were stimulated with LPA (50 μM) for 8 h. No.4–6 samples were pre-treated with Y-27632 (10 μM) before stimulation with LPA (50 μM) for 8 h. Heat map depicts the relative expression values for the 37 cytokines ( n = 3). ( B ) HAECs were treated with Y-27632 (10 μM) for 30 min and then were stimulated by LPA (50 μM) for 12 h. Culture media were harvested and followed by ELISA ( n = 3). * p < 0.05. ( C ) HAECs were pre-treated with Y-27632 (10 μM) before stimulation with LPA (50 μM) for 4 h. MCP-1 mRNA was analysed by quantitative real-time PCR ( n = 3). * p < 0.05. ( D ) HAECs were pre-treated with Y-27632 (10 μM) and then stimulated with LPA (50 μM) for 8 h. Cell lysates were subjected to Western blot analysis for E-selectin. β-actin was loaded as internal control. The histogram shows the relative intensity of each band ( n = 3). * p <0.05. ( E ) HAECs were pre-treated with Y-27632 (10 μM) before stimulation with LPA (50 μM) for 8 h. E-selectin mRNA was analysed by quantitative real-time PCR ( n = 3). * p < 0.05. ( F ) HAECs were transfected with a pGL3-ELAM-LUC construct. Cells were pre-treated with Y-27632 (10 μM) before stimulation with LPA (50 μM) for 4 h. The bar graph shows the relative luciferase activity of each sample ( n = 3). * p < 0.05. Data are expressed as means ± SEM.

Article Snippet: Human aortic endothelial cells (HAECs) (Cat# C-12271) and THP-1 (Cat# TIB202) were purchased from PromoCell (Heidelberg, Germany) and ATCC (Manassas, VA, USA) respectively but were not further authenticated after purchase.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Control, Transfection, Construct, Luciferase, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: ROCK2 Regulates Monocyte Migration and Cell to Cell Adhesion in Vascular Endothelial Cells

doi: 10.3390/ijms20061331

Figure Lengend Snippet: Kinetics of IκBα is under the control of ROCK signalling. ( A ) HAECs were pre-treated with Bay 11-7082 (5 μM) before stimulation with LPA (4 h). MCP-1 and E-selectin mRNA expression levels were analysed by quantitative real-time PCR ( n = 3). * p < 0.05 vs. LPA alone. ( B ) HAECs were pre-treated with Y-27632 (10 μM) before stimulation with LPA (50 μM) for 1 h. Cell lysates were prepared and assayed for IκBα phosphorylation by Western blot analysis. A representative blot of three independent experiments is shown. The bottom histogram shows the relative intensity of each band. * p < 0.05. ( C ) HAECs were pre-treated with Y-27632 (10 μM) before LPA (50 μM) stimulation for 1 h. Cell lysates were prepared and assayed for IκBα expression and RelA/p65 phosphorylation by Western blot analysis. A representative blot of three independent experiments is shown. The histograms show the relative intensity of each band. * p < 0.05. ( D ) HAECs were pre-treated with Y-27632 (10 μM) and then stimulated with LPA (50 μM) for 1 h. Cytoplasmic and nuclear extracts were prepared and assayed for nuclear translocation of RelA/p65 by Western blot analysis. A representative blot of three independent experiments is shown. The histograms show the relative intensity of each band. * p < 0.05. ( E ) HAECs were treated with LPA (50 μM) for 1 h. In a set of experiments, cells are pre-treated with Y-27632 (10 μM). Cells were fixed and stained with anti-RelA/p65 antibody (red) and Hoechst (blue) (magnification ×400). A representative photomicrograph of three independent experiments is shown. Scale bar, 10 μm. Data are expressed as means ± SEM.

Article Snippet: Human aortic endothelial cells (HAECs) (Cat# C-12271) and THP-1 (Cat# TIB202) were purchased from PromoCell (Heidelberg, Germany) and ATCC (Manassas, VA, USA) respectively but were not further authenticated after purchase.

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Translocation Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: ROCK2 Regulates Monocyte Migration and Cell to Cell Adhesion in Vascular Endothelial Cells

doi: 10.3390/ijms20061331

Figure Lengend Snippet: LPA-induced MCP-1 and E-selectin expression is possibly mediated via ROCK2 in HAECs. ( A ) HAECs were stimulated with LPA (50 μM) for 5 min. Cell lysates were subjected to immunoprecipitation analysis for detecting ROCK1 (left panel) or ROCK2 (right panel) activity. A representative blot of three independent experiments is shown. Each histogram shows the ratio of each band. * p < 0.05. ( B ) HAECs were treated with scrambled control siRNA or Rho-kinase isoform specific siRNA. Relative mRNA levels of ROCK1 (left panel) and ROCK2 (right panel) were analysed by quantitative real-time PCR ( n = 3). * p < 0.05. ( C ) HAECs were treated with scrambled control siRNA or Rho-kinase isoform specific siRNA. Cell lysates were prepared and assayed for ROCK1 and ROCK2 by Western blot analysis. A representative blot of three independent experiments is shown. Each histogram shows the ratio of each band. * p < 0.05. ( D , E ) HAECs stimulated with LPA (50 μM) for 4h or 8 h were treated with scrambled control siRNA or Rho-kinase isoform specific siRNA and relative mRNA levels of MCP-1 ( D ) and E-selectin ( E ) were analysed by quantitative real-time PCR ( n = 3). * p < 0.05. Data are expressed as means ± SEM.

Article Snippet: Human aortic endothelial cells (HAECs) (Cat# C-12271) and THP-1 (Cat# TIB202) were purchased from PromoCell (Heidelberg, Germany) and ATCC (Manassas, VA, USA) respectively but were not further authenticated after purchase.

Techniques: Expressing, Immunoprecipitation, Activity Assay, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: ROCK2 Regulates Monocyte Migration and Cell to Cell Adhesion in Vascular Endothelial Cells

doi: 10.3390/ijms20061331

Figure Lengend Snippet: Endothelial ROCK2 regulates recruitment of monocytic cells. ( A ) The principle of migration assay. ( B ) HAECs were treated with scrambled control siRNA or MCP-1 siRNA. Relative mRNA levels of MCP-1 were analysed by quantitative real-time PCR ( n = 3). * p < 0.05. ( C ) HAECs were treated with scrambled control siRNA or MCP-1 siRNA. Culture media were harvested and MCP-1 protein levels were analysed by ELISA ( n = 3). * p < 0.05. ( D , E ) HAECs stimulated with LPA (50 μM) for 12 h were treated with scrambled control siRNA, Rho-kinase isoform specific siRNA or MCP-1 siRNA. The HAECs supernatants were then collected and assayed for their chemotactic activity on THP-1 cells through Transwell chemotaxis chamber. The migrated THP-1 cells were observed with the use of light microscopy (magnification ×100) ( D ) and the migrated cells were counted ( E ) ( n = 3). * p < 0.05. Data are expressed as means ± SEM.

Article Snippet: Human aortic endothelial cells (HAECs) (Cat# C-12271) and THP-1 (Cat# TIB202) were purchased from PromoCell (Heidelberg, Germany) and ATCC (Manassas, VA, USA) respectively but were not further authenticated after purchase.

Techniques: Migration, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activity Assay, Chemotaxis Assay, Light Microscopy

Journal: International Journal of Molecular Sciences

Article Title: ROCK2 Regulates Monocyte Migration and Cell to Cell Adhesion in Vascular Endothelial Cells

doi: 10.3390/ijms20061331

Figure Lengend Snippet: Endothelial ROCK2 regulates cell to cell adhesion. ( A ) The principle of adhesion assay. ( B ) HAECs were treated with scrambled control siRNA or E-selectin siRNA. Relative mRNA levels of E-selectin were analysed by quantitative real-time PCR ( n = 3). * p < 0.05. ( C ) HAECs were treated with scrambled control siRNA or E-selectin siRNA. Cell lysates were prepared and assayed for E-selectin by Western blot analysis. A representative blot of three independent experiments is shown. The bottom histogram shows the ratio of each band. * p < 0.05. ( D ) HAECs stimulated with LPA (50 μM) for 8 h were treated with scrambled control siRNA or Rho-kinase isoform specific siRNA. Cell lysates were analysed for cell adhesion to THP-1 using adhesion assay. The bar graph shows the relative fluorescence unit of each sample ( n = 3). * p < 0.05. Data are expressed as means ± SEM.

Article Snippet: Human aortic endothelial cells (HAECs) (Cat# C-12271) and THP-1 (Cat# TIB202) were purchased from PromoCell (Heidelberg, Germany) and ATCC (Manassas, VA, USA) respectively but were not further authenticated after purchase.

Techniques: Cell Adhesion Assay, Control, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: ROCK2 Regulates Monocyte Migration and Cell to Cell Adhesion in Vascular Endothelial Cells

doi: 10.3390/ijms20061331

Figure Lengend Snippet: Mechanism of ROCK2-mediated cell to cell adhesion and monocyte migration in HAECs. In vascular endothelial cells, LPA induces selective activation ROCK2 and expression of chemokines and E-selectin. As a result, cell to cell adhesion and monocyte migration are increased.

Article Snippet: Human aortic endothelial cells (HAECs) (Cat# C-12271) and THP-1 (Cat# TIB202) were purchased from PromoCell (Heidelberg, Germany) and ATCC (Manassas, VA, USA) respectively but were not further authenticated after purchase.

Techniques: Migration, Activation Assay, Expressing