Journal:

Article Title: Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1 (VEGFR-1)

doi: 10.1016/j.devcel.2009.07.011

Figure Lengend Snippet: (A-B) Confocal images of blood vessels from PBS- or Flt-1 neutralizing antibody-injected P5 mouse retinas stained with Alexa488-conjugated isolectin-B4. Arrowheads denote filopodia extended either in the direction of migration (A), or toward the sprout base (B). (C-F) Vessel sprouts were analyzed for guidance parameters (D: * = p≤0.01; E and F: *** = p≤0.0002). (F) Values are averages + SD. (G-H) Confocal images of blood vessels from P6 mouse retinas injected with adenovirus expressing either GFP (Ad-GFP) or cre-recombinase (Ad-Cre) on P3, and stained with Alexa488-conjugated isolectin-B4. Arrowheads denote filopodia extended either in the direction of migration (G), or toward the sprout base (H). (I-L) Vessel sprouts were analyzed for guidance parameters (K: ** = p≤0.002; L: *** = p≤0.0001). (L) Values are averages + SD. Scale Bar, 20 μm.

Article Snippet: Flt-1 flox/flox (Genentech) and flt-1 flox/+ mouse pups (P3) were anesthetized, and approximately 0.5 μl (1×10 10 PFU/ml) of GFP- or Cre-expressing adenovirus was injected into each eye.

Techniques: Injection, Staining, Migration, Expressing

Journal: Acta Pharmacologica Sinica

Article Title: ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation

doi: 10.1038/s41401-018-0072-0

Figure Lengend Snippet: Effects of ClC-3 on Ang II-induced ROS production in HUVECs. a Ang II treatment increased ClC-3 expression in HUVECs (*P < 0.05 vs. CON. n = 5). b and c Effects of adenovirus (Ad)-ClC-3 shRNA and Ad-ClC-3 transfection on the expression of ClC-3 in HUVECs. Cells were transfected with Ad-ClC-3 shRNA (100 MOI) (b) or Ad-ClC-3 (100 MOI) (c) for 48 h (*P < 0.05 vs. CON. n = 4). d and e Representative images (× 400) with quantitative analysis of the dihydroethidium (DHE) fluorescence intensity (in arbitrary units) were shown in HUVECs transfected with Ad-ClC-3 shRNA (d) or Ad-ClC-3 (e) upon stimulation with Ang II (1 μmol/L) for 24 h. Ang II-induced ROS production was attenuated by knockdown of ClC-3, and further enhanced by ClC-3 overexpression (n = 6, **P < 0.01 vs. CON, ##P < 0.01 vs. Ang II). f ClC-3 potentiated Ang II-induced senescence of HUVECs. HUVECs were transfected with Ad-ClC-3 shRNA or Ad-ClC-3 for 48 h and then were incubated with or without Ang II (1 μmol/L) for 24 h. Representative photomicrographs (× 400) show the senescence-associated β-galactosidase (SA-β-Gal)-positive cells with different treatments (n = 6)

Article Snippet: Adenovirus transfection Adenovirus (Ad)-ClC-3 shRNA and Ad-ClC-3 cDNA were designed and produced by Sunbio Medical Biotechnology (Shanghai, China).

Techniques: Expressing, shRNA, Transfection, Fluorescence, Knockdown, Over Expression, Incubation

Journal: Acta Pharmacologica Sinica

Article Title: ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation

doi: 10.1038/s41401-018-0072-0

Figure Lengend Snippet: ClC-3 increased p47phox phosphorylation through p38 MAPK pathway. a The time course of p47phox and p38 MAPK phosphorylation in Ang II-treated HUVECs (n = 5, *P < 0.05 vs. CON, **P < 0.01 vs. CON). b Knockdown of ClC-3 decreased p38 MAPK phosphorylation induced by Ang II (1 μmol/L) treatment for 30 min. c Overexpression of ClC-3 enhanced p38 MAPK phosphorylation (n = 5, *P < 0.05 vs. CON, #P < 0.05 vs. Ang II). d–f Cells were pretreated with the p38 MAPK inhibitor SB203580 (10 μmol/L) for 6 h before Ad-ClC-3 transfection. SB203580 abolished the increase in p47phox phosphorylation (d), NADPH oxidase activity (e), and ROS generation (f) induced by ClC-3 overexpression in HUVECs with Ang II treatment, respectively (n = 6, **P < 0.01 vs. Ang II, ##P < 0.01 vs. Ang II+Ad-ClC-3)

Article Snippet: Adenovirus transfection Adenovirus (Ad)-ClC-3 shRNA and Ad-ClC-3 cDNA were designed and produced by Sunbio Medical Biotechnology (Shanghai, China).

Techniques: Phospho-proteomics, Knockdown, Over Expression, Transfection, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Rac1 and RhoG activation upon ICAM-1 clustering requires filamin. (A) Formation of F-actin–positive docking structures around transmigrating neutrophils was visualized by GFP-LifeAct in TNF-α–stimulated HUVEC. Top, left, GFP-LifeAct recruitment at different Z-stack levels, as indicated above. Bottom, left, a docking structure along the x–z axis. The leukocyte is marked with a asterisk. Middle, a differential interference contrast (DIC) image of a transmigrating neutrophils (XY, asterisk). Right, a three-dimensional Z-stack projection (3D). (B) For assessment of filamin C interaction with ICAM-1, HUVEC were transfected with control or filamin A and B siRNA, and pulldown assays were performed with the ICAM-1 C-terminal peptide. Streptavidin agarose without peptide was used as a control. (C) siRNA-mediated knockdown of filamin A/B/C expression in HUVEC was confirmed by Western blotting. Knockdown of filamin does not affect ICAM-1 expression induced by 20-h TNF-α treatment. Actin was used as a control for equal sample loading. ICAM-1 was clustered (CL) with α-ICAM-1–antibody beads on siCtrl and siFilamin A/B/C-transfected HUVEC for 10 and 30 min, and Rac1-GTP (D) and RhoG-GTP (E) were precipitated with biotinylated Pak1-CRIB peptide coupled to streptavidin agarose and GST-ELMO coupled to glutathione Sepharose pulldown (PD) assays, respectively. Rac1.GTP and RhoG.GTP levels were quantified and corrected for Rac1 and RhoG total lysate levels with ImageJ. Quantification of Western blots represents an average of at least three independent experiments ± SEM. *, p < 0.05.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Activation Assay, Transfection, Expressing, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: ICAM-1 clustering induces activation of the GEF Trio. (A) Trio (350 kDa) and Vav2 (100 kDa) protein expression in HUVEC (HUVEC, lane 1; HUVEC treated for 20 h with TNF-α, lane 2), human brain endothelial cells (hCMEC/D3, lane 3), and HMVEC (lane 4). Trio (B and D) and Vav2 (C and E) activation was assessed by their affinity in pulldown (PD) assays for GST-fusion protein of nucleotide-free mutants of Rac1 G15A (B and C) and RhoG G15A (D and E) coupled to glutathione Sepharose. Trio and Vav2 binding were quantified and corrected for Trio and Vav2 total lysate levels with ImageJ. Quantification of Western blots represents an average of at least three independent experiments ± SEM. *, p < 0.05.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Activation Assay, Expressing, Binding Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Trio and TrioD1 colocalize with ICAM-1 upon clustering. Endogenous ICAM-1 (red) in TNF-α–stimulated endothelial cells colocalizes with GFP-Trio full length to 10-μm α-ICAM-1–antibody beads (A, green) or to adherent neutrophils (B) and with TrioD1 (C, green) to these beads (asterisks). In (B), nuclei of neutrophils are visualized with DAPI staining and are included in the DIC images. Scale bars: 10 μm.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Staining

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Trio interacts with ICAM-1 independently of filamin. (A) Cos7 cells were transiently transfected with Myc-TrioD1 (B), and pulldown assays were performed with the ICAM-1 C-terminal peptide, streptavidin-agarose without peptide (CTRL), or a peptide of the VCAM-1 C-terminal intracellular tail, as described in Materials and Methods . Interaction was assessed by immunoblotting with anti-Myc antibody. Filamin A binding to the ICAM-1 C-terminal peptide was used as a positive control. (B) HUVEC were transfected with siRNA targeting filamin A/B, filamin C, or filamin A/B/C together, and pulldown assays were performed with the ICAM-1 C-terminal peptide for endogenous Trio. No difference in binding of Trio to the ICAM-1 C-terminal peptide was observed. Actin was used as protein loading control. A representative result of three independent experiments is shown. Quantification is shown in Figure S3B.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Transfection, Western Blot, Binding Assay, Positive Control

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Trio activation upon ICAM-1 clustering requires filamin. ICAM-1 was clustered (CL) for 10 and 30 min on TNF-α–stimulated HUVEC transfected with siRNA targeting filamin A/B/C. Trio activation was assessed by its affinity in pulldown (PD) assays with a GST-fusion protein of nucleotide-free mutant of Rac1 G15A (A) or RhoG G15A (B) coupled to glutathione Sepharose. Trio binding was quantified and corrected for Trio total lysate levels with ImageJ. Quantification of Trio binding to GST-Rac1 G15A Western blots represents an average of three independent experiments ± SEM.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Activation Assay, Transfection, Mutagenesis, Binding Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Trio knockdown or inhibition reduces neutrophil adhesion and TEM. HUVEC were transduced with shTrio-expressing lentivirus to silence Trio expression. (A) Knockdown of Trio expression was confirmed by Western blot. β-Catenin and actin were used as a control for equal loading. Neutrophil adhesion to and TEM across (B) shTrio-transduced and TNF-α–stimulated HUVEC was measured under physiological flow conditions as described in Materials and Methods . Data are means of three independent experiments ± SEM. (C) ICAM-1 expression in shTrio-transduced and TNF-α–stimulated HUVEC was analyzed by Western blotting. Actin was used as a control for equal loading. (D) TrioN was expressed using adenovirus into endothelial cells that were or were not silenced for Trio (shTrio). TEM assay under flow conditions was performed and showed that TrioN rescued impaired neutrophil TEM across Trio-deficient endothelial cells. The experiment was done twice in triplicate. Data are means ± SEM. (E and F) TNF-α–stimulated HUVEC were grown to confluency, treated with 100 μM ITX3 for 16 h or DMSO as a control. The resistance or confluency was not affected by the treatments. Neutrophil adhesion (E) and TEM (F) were measured under physiological flow conditions as described in Materials and Methods . Data are means of three independent experiments ± SEM. (G) HUVEC were microporated with GFP-LifeAct to visualize F-actin during live-cell imaging. TNF-α–stimulated HUVEC were treated with 100 μM ITX3 (lower panels) for 16 h or dimethyl sulfoxide (DMSO) as a control (top panels) and neutrophils were allowed to transmigrate through endothelial monolayers. The focal plane was set at the surface of the endothelium in ITX3-treated cells, since in contrast to controls cells (DMSO), no actin-positive rings are observed in the upper focal plane. Neutrophils are visualized in DIC panels. “XZ” panels show Z-stack reconstructions of endothelial docking structures. Asterisk indicates transmigrating neutrophils. (H) Quantification of GFP-LifeAct–positive docking structures around transmigrating neutrophils. Data are means of three independent experiments ± SEM. *, p < 0.05; ***, p < 0.001. Scale bar: 10 μm.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Inhibition, Transduction, Expressing, Western Blot, Live Cell Imaging

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Trio inhibition with ITX3 impairs endothelial docking structure formation. (A) ICAM-1 was clustered with 10-μm α-ICAM-1–antibody beads on TNF-α–stimulated HUVEC were treated with 100 μM ITX3 for 16 h or DMSO as a control. ICAM-1 and F-actin localization were analyzed by immunofluorescence. Beads are visualized in DIC panels and indicated by arrowheads. (B) TNF-α–stimulated HUVEC transduced with ICAM-1-GFP adenovirus and treated with ITX3 or DMSO as a control were imaged live, and continuous and discontinuous rings around α-ICAM-1–antibody beads were quantified after 30 min of ICAM-1 clustering. Rings were quantified around > 50 beads per experiment. Data are means of eight experiments ± SEM. (C) F-actin ring quantification. F-actin rings were quantified around > 50 beads per experiment. Data are means of four independent experiments ± SEM. (D) ICAM-1 ring diameter in TNF-α–stimulated HUVEC transduced with ICAM-1-GFP adenovirus and treated with ITX3 or DMSO as a control. ICAM-1 is shown in green and the bead in red. (E) ICAM-1 ring diameter quantification. ICAM-1 ring diameter was measured for ∼30 beads per experiment. Data are means of eight experiments ± SEM. (F) Reconstruction of Z-stack imaging in TNF-α–stimulated HUVEC transduced with ICAM-1-GFP adenovirus and treated with ITX3 or DMSO as a control shows ICAM-1–positive docking structure around a α-ICAM-1–antibody bead. Average length of ICAM-1–positive membrane protrusions around ∼30 α-ICAM-1–antibody beads per experiment was measured in TNF-α–stimulated HUVEC treated with ITX3 (G) or with Rac1 inhibitor NSC23766 (H). Data are means of three independent experiments ± SEM. (I). Schematic representation of the effect of Trio inhibition by ITX3 on endothelial docking structure formation. *, p < 0.05; **, p < 0.01. Scale bars: 10 μm.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Inhibition, Immunofluorescence, Transduction, Imaging

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Sequential Rac1 and RhoG activation upon ICAM-1 clustering. (A) ICAM-1 was clustered (CL) with α-ICAM-1–antibody beads for indicated time points, and Rac1-GTP and RhoG-GTP were precipitated as described in Materials and Methods . ICAM-1 is shown as loading control. Graph on the right shows quantification of the blots by ImageJ. Dotted line represents active Rac1 levels (square) and filled line represents active RhoG levels (circle). Data are mean of three independent experiments ± SEM. (B) Graphs show quantification of active Rac1 levels (left) or active RhoG levels (right) upon ICAM-1 clustering. ITX3 inhibits ICAM-1–induced Rac1 and RhoG activation. Data are an average of three independent experiments ± SEM.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Activation Assay

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Trio mediates endothelial docking structure formation through Rac1 and RhoG. (A) To silence Rac1 and RhoG expression, we used lentivirally delivered constructs targeting Rac1 and RhoG mRNA in TNF-α–stimulated HUVEC. Knockdown of Rac1 and RhoG expression was confirmed by Western blotting and did not affect ICAM-1 expression. Tubulin or actin was used as loading control. (B) Average length of ICAM-1–positive membrane protrusions around ∼30 α-ICAM-1–antibody beads per experiment was quantified. Data are means of three independent experiments ± SEM. (C) ICAM-1 was clustered with 10-μm α-ICAM-1–antibody beads on TNF-α–stimulated HUVEC with silenced Rac1 or RhoG expression. ICAM-1 and F-actin localization were analyzed by immunofluorescence. Beads are visualized in DIC panels and indicated by arrowheads. Scale bars: 10 μm. (D) Neutrophils were allowed to adhere to TNF-α–stimulated HUVEC silenced for Rac1 or RhoG. x–z projections show the absence of ICAM-1–positive protrusions upon Rac1 or RhoG silencing. Scale bars: 20 μm. (E) Neutrophil TEM were significantly impaired upon Rac1 or RhoG silencing in TNF-α–stimulated HUVEC. Data are means of three independent experiments ± SEM. *, p < 0.01.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Expressing, Construct, Western Blot, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

doi: 10.1091/mbc.E11-11-0907

Figure Lengend Snippet: Model for ICAM-1–induced endothelial docking structure formation. Clustering of ICAM-1 induces the recruitment of the F-actin cross-linker protein filamin. Filamin functions as a scaffold for subsequent activation of the GEF Trio. In turn, Trio activates the GTPase Rac1 through its N-terminal GEF domain, leading to the recruitment of other adapter proteins, such as cortactin and α-actinin-1/4. ICAM-1 clustering also induces the activation of the GTPase RhoG through Trio and SGEF. On their activation, Rac1 and RhoG induce remodeling of the F-actin cytoskeleton, leading to endothelial docking structure formation. Formation of docking structures serves to guide adherent leukocytes during diapedesis. Image is not drawn to scale.

Article Snippet: Adenovirus expressing ICAM-1-GFP or GFP-TrioD1 was produced by transfecting Pac I-digested (Westburg, Leusden) constructs into HEK293T cells.

Techniques: Activation Assay