huvec nucleofector solution  (Amaxa)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

doi: 10.1161/JAHA.116.003336

Figure Lengend Snippet: Rnd3 in the microcirculation and in cultured endothelial cells. A, Widefield images of a rat mesenteric window labeled with anti‐Rnd3 (top) and anti‐ VE ‐cadherin antibodies (bottom), showing a relatively high degree of Rnd3 labeling in microvessels. B, Widefield image of HUVEC labeled with anti‐Rnd3 antibodies, revealing strong patches of Rnd3 labeling adjacent to the nuclei (large arrows), and tubule‐like labeling reaching into the cytoplasm (small arrows). C, Single confocal slice images of Rnd3 labeling (left), with counter‐labeling for the trans‐Golgi network with TGN 46 (middle), in HUVEC . The overlay image shows that a portion of the intense areas of Rnd3 labeling near the nuclei are within the trans‐Golgi network. Images are representative of at least 3 separate labeling experiments. These labeling patterns were not observed in control experiments with no primary antibody (not shown). HUVEC indicates human umbilical vein endothelial cells.

Article Snippet: The number of cells was counted, the suspension was centrifuged at 100 g for 10 minutes, and the pellet was resuspended in HUVEC Nucleofector solution (Lonza) at 5×10 6 cells/mL.

Techniques: Cell Culture, Labeling, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

doi: 10.1161/JAHA.116.003336

Figure Lengend Snippet: Impact of specific Rnd3 depletion on thrombin‐induced endothelial barrier dysfunction. A, Concentration‐dependent impact of Rnd3 si RNA on Rnd3 expression in HUVEC vs si CONTROL nontargeting RNA (Ctrl). RhoA and β‐tubulin served as loading controls. Blots are representative of 3 experiments. B, Specific Rnd3 depletion with si RNA prolongs thrombin‐induced decreases in HUVEC monolayer TER . C, Rnd3 depletion also prolongs thrombin‐induced increases in P s albumin . § P <0.05, §§ P <0.01 vs baseline. * P <0.05 vs si CONTROL , same time point. HUVEC indicates human umbilical vein endothelial cells; TER, transendothelial electrical resistance.

Article Snippet: The number of cells was counted, the suspension was centrifuged at 100 g for 10 minutes, and the pellet was resuspended in HUVEC Nucleofector solution (Lonza) at 5×10 6 cells/mL.

Techniques: Concentration Assay, Expressing, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

doi: 10.1161/JAHA.116.003336

Figure Lengend Snippet: Rnd3 overexpression attenuates thrombin‐induced endothelial barrier dysfunction. A, Western blot showing overexpression of MAT ‐ FLAG ‐Rnd3 in HUVEC . B, Immunofluorescence image showing very high expression of MAT ‐ FLAG ‐Rnd3 (green) throughout the cytoplasm in 2 transfected cells. Nuclei are labeled blue. The large arrows show areas of high expression in the perinuclear area, similar to those seen with endogenous Rnd3. The small arrows show accumulation at the cell borders and cell–cell contacts. C, Rnd3 overexpression in HUVEC attenuates the thrombin‐induced drop in TER and increase in P s albumin (D). * P <0.05, ** P <0.01 between groups at the same time point. § P <0.05, §§ P <0.01 vs baseline. HUVEC indicates human umbilical vein endothelial cells; IB, immunoblot; TER, transendothelial electrical resistance.

Article Snippet: The number of cells was counted, the suspension was centrifuged at 100 g for 10 minutes, and the pellet was resuspended in HUVEC Nucleofector solution (Lonza) at 5×10 6 cells/mL.

Techniques: Over Expression, Western Blot, Immunofluorescence, Expressing, Transfection, Labeling

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

doi: 10.1161/JAHA.116.003336

Figure Lengend Snippet: Impact of Rnd3 overexpression or depletion on RhoA, Rac1, and Cdc42 activation by thrombin. A, RhoA‐ GTP activation in mock‐transfected or MAT ‐ FLAG ‐Rnd3‐transfected HUVEC monolayers during baseline conditions and 5, 30, 60, and 120 minutes following the addition of 1 U/mL thrombin. B and C, Rac1 and Cdc42 activation, respectively, under the same conditions. D, RhoA activation in HUVEC treated with Rnd3 si RNA or si CONTROL nontargeting RNA under baseline conditions and after treatment with 1 U/mL thrombin for 5, 30, 60, or 120 minutes. E and F, Activation of Rac1 and Cdc42 under the same conditions. * P <0.05 vs baseline. † P <0.05 vs. MAT‐FLAG‐Rnd3, same time point. § P <0.05 between groups, same time point. The numbers in parentheses at each time point indicate the number of replicates for the mock and MAT ‐ FLAG ‐Rnd3 groups, respectively, in (A through C) and for the si CONTROL and Rnd3 si RNA groups, respectively, in (D through F). HUVEC indicates human umbilical vein endothelial cells.

Article Snippet: The number of cells was counted, the suspension was centrifuged at 100 g for 10 minutes, and the pellet was resuspended in HUVEC Nucleofector solution (Lonza) at 5×10 6 cells/mL.

Techniques: Over Expression, Activation Assay, Transfection, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

doi: 10.1161/JAHA.116.003336

Figure Lengend Snippet: Rnd3 overexpression limits thrombin‐induced actin cytoskeletal changes and endothelial cell retraction. HUVEC expressing GFP ‐actin alone (Control) or in combination with mCherry‐Rnd3 were used for these experiments. A, The protrusion frequency of local lamellipodia is shown for baseline ( BL ) and at 10‐minute intervals after the addition of 1 U/mL thrombin. * P <0.05 and ** P <0.01 vs baseline, same treatment group. B, In the same groups of cells, actin fibers were counted at baseline and after addition of thrombin. The mean peak number of fibers after the addition of thrombin is shown. † P <0.05 vs baseline for the nontransfected control group. C, Changes in the ventral surface area covered by each cell were determined at baseline, and 30 and 60 minutes after the addition of thrombin. Data normalized to the mean at baseline for each group are shown. § P <0.05 mC herry‐Rnd3 vs control, same time point. N=6 cells in each group. HUVEC indicates human umbilical vein endothelial cells.

Article Snippet: The number of cells was counted, the suspension was centrifuged at 100 g for 10 minutes, and the pellet was resuspended in HUVEC Nucleofector solution (Lonza) at 5×10 6 cells/mL.

Techniques: Over Expression, Expressing, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

doi: 10.1161/JAHA.116.003336

Figure Lengend Snippet: Baseline TER of Untransfected HUVEC Monolayers Versus Those Transfected With Rnd3 siRNA, siCONTROL RNA, MAT‐FLAG‐Rnd3, or no Vector/siRNA (Mock)

Article Snippet: The number of cells was counted, the suspension was centrifuged at 100 g for 10 minutes, and the pellet was resuspended in HUVEC Nucleofector solution (Lonza) at 5×10 6 cells/mL.

Techniques: Transfection, Plasmid Preparation

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

doi: 10.1161/JAHA.116.003336

Figure Lengend Snippet: Baseline P s albumin of Untransfected HUVEC Monolayers Versus Those Transfected With Rnd3 siRNA, siCONTROL RNA, MAT‐FLAG‐Rnd3, or no Vector/siRNA (Mock)

Article Snippet: The number of cells was counted, the suspension was centrifuged at 100 g for 10 minutes, and the pellet was resuspended in HUVEC Nucleofector solution (Lonza) at 5×10 6 cells/mL.

Techniques: Transfection, Plasmid Preparation

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) upregulates vascular endothelial growth factor (VEGF)-C expression in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent manner. (A) HUVECs were incubated with S1P for 4 h at the indicated concentrations. RNAs were harvested and analyzed by real-time PCR using specific primer sets for human VEGF-C or GAPDH. (B) HUVECs were incubated with S1P (5 μmol/L) for different time intervals as indicated and subjected to real-time PCR. (C) VEGF-C levels in the supernatant of cultured HUVECs were measured by ELISA after incubation with S1P for 8 h at various concentrations as indicated. (D) HUVECs were incubated with S1P (5 μmol/L) for the indicated time intervals and VEGF-C levels in the supernatant were measured by ELISA. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) upregulates vascular endothelial growth factor (VEGF)-C expression by a matrix metalloproteinase (MMP)-2-dependent pathway in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were pretreated with GM6001 (10 μmol/L) for 1 h, followed by S1P (5 μmol/L) treatment for another 4 h. VEGF-C mRNA expression levels were monitored by real-time PCR. (B) siRNA mediated downregulation of MMP-2 mRNA expression in HUVECs. MMP-2 and scrambled siRNA were transfected into HUVECs by electroporation. The transfection efficiency was evaluated by real-time PCR. MMP-2 expression was suppressed in HUVECs transiently transfected with MMP-2 siRNA. (C) HUVECs transfected with scrambled or MMP-2 siRNA were incubated with S1P (5 μmol/L) for 4 h, and VEGF-C mRNA expression levels were monitored by real-time PCR. (D) HUVECs were incubated with S1P (5 μmol/L) for 8 h, and the secreted concentration of VEGF-C protein was determined by ELISA. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Electroporation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) upregulates vascular endothelial growth factor (VEGF)-C expression by a fibroblast growth factor receptor (FGFR)-1-dependent pathway in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were pretreated with SU5402 (5 μmol/L) or AG1478 (100 nmol/L) for 1 h, followed by S1P (5 μmol/L) treatment for another 4 h. VEGF-C mRNA expression levels were monitored by real-time PCR. (B) Total mRNA was extracted from HUVECs, and reverse-transcribed to cDNA. According to real-time PCR, the relative levels of different FGF receptors normalized to GAPDH are shown. (C) FGFR-1 and scrambled siRNA were transfected into HUVECs by electroporation. The transfection efficiency was evaluated by real-time PCR. FGFR-1 expression was suppressed in HUVECs transiently transfected with FGFR-1 siRNA. (D) HUVECs were transfected with scrambled or FGFR-1 siRNA, followed by incubation with S1P (5 μmol/L) for 4 h, and VEGF-C mRNA expression levels were monitored by real-time PCR. (E) HUVECs were incubated with S1P (5 μmol/L) for 8 h, and the secreted concentration of VEGF-C protein was determined by ELISA. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control. fP<0.01 vs S1P.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription, Transfection, Electroporation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P) induces fibroblast growth factor (FGF) receptor (FGFR)-1 phosphorylation in a time-dependent manner in human umbilical vein endothelial cells (HUVECs). (A) Starved HUVECs were treated with S1P (5 μmol/L) for 1 and 5 min. The concentration of phosphorylated FGFR-1 was measured by an FGFR-1-phospho ELISA kit. (B) HUVECs were pretreated with GM6001 (5 μmol/L) for 1 h, followed by S1P or FGF-2 treatment for 1 min. The concentration of phosphorylated FGFR-1 was measured by an FGFR-1-phospho ELISA kit. All ELISA data are expressed as the mean±SD from at least three independent experiments. bP<0.05 vs control. eP<0.05 vs S1P.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Phospho-proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Acta Pharmacologica Sinica

Article Title: Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

doi: 10.1038/aps.2012.186

Figure Lengend Snippet: Sphingosine-1-phosphate (S1P)-upregulated vascular endothelial growth factor (VEGF)-C mRNA expression is suppressed by shRNA of fibroblast growth factor (FGF)-1 but not siRNA of FGF-2 in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with scrambled, FGF-1 shRNA (A), and FGF-2 siRNA (B) by electroporation, and the transfection efficiency was evaluated by real-time PCR. FGF-1 (C) and FGF-2 knockdown (D) HUVECs were incubated with S1P (5 μmol/L) for 4 h, and VEGF-C mRNA expression levels were monitored by real-time PCR. Quantified results are shown as the mean±SD from at least three independent experiments. bP<0.05, cP<0.01 vs control.

Article Snippet: Cells (5×10 5 ) were resuspended in 100 μL of supplemented HUVEC Nucleofector TM solution (Amaxa Biosystems) and electroporated with 30 pmol siRNA or shRNA.

Techniques: Expressing, shRNA, Transfection, Electroporation, Real-time Polymerase Chain Reaction, Knockdown, Incubation, Control