44 research outputs found

    HIV-1 gp120 induces hyperpermeability in L-LECs through activation of α<sub>5</sub>β<sub>1</sub> integrin.

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    <p>(<b>A</b>) Representative Western blot analysis of phosphorylated α<sub>5</sub>β<sub>1</sub> integrin in L-LECs after 2 hours of serum starvation and subsequent incubation for designated times with HIV-1 gp120 (500 ng/ml) or Slit2N (500 ng/ml). GAPDH used as loading control. (<b>B</b>) Phosphorylated α<sub>5</sub>β<sub>1</sub> integrin and HIV-1 gp120 expression and co-localization in L-LECs by confocal microscopy. L-LECs were cultured in chamber slides and incubated with HIV-1 gp120 (500 ng/ml) for 15 minutes before fixing and staining cells. Scale bars = 10 µm. (<b>C</b>) Permeability through an L-LEC monolayer as previously described. L-LEC monolayers were pretreated with a neutralizing anti-integrin β<sub>1</sub> antibody or a normal IgG control for 2 hours before incubating with M-gp120 or T-gp120 (both 500 ng/ml) for 18 hours. Data indicate the mean ± SD of 3 independent experiments. (**p<0.01; *** p<0.001).</p

    The fibronectin domains of Robo4 are critical for gp120-induced hyperpermeability of L-LEC monolayers.

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    <p>(<b>A</b>) Representative Western blot analysis of phosphorylated c-Src in L-LECs pretreated with Slit2N (500 ng/ml) or a control for 1 hour, then stimulated with fibronectin (FN) [1 µg/ml (1×); 10 µg/ml (10×)] for 20 minutes as indicated. GAPDH used as loading control. (<b>B</b>) Permeability through an L-LEC monolayer as previously described. L-LECs were transiently transfected with expression plasmids encoding wild-type Robo4 (WT), mutant Robo4 (MT), or a vector control (V). After 48 hours, cells were plated for the permeability assay per manufacturer's instructions. L-LEC monolayers were incubated overnight with 500 ng/ml HIV-1 gp120 or a control. Data are represented as the percentage increase in permeability of each cell type monolayer incubated with gp120 vs. control. Data indicate the mean ± SD of 3 independent experiments. (*p<0.05, ***p<0.001). (<b>C</b>) Representative Western blot analysis of phosphorylated c-Src and ERK1/2 in L-LECs transiently transfected with expression plasmids encoding wild-type Robo4 (WT), mutant Robo4 (MT), or a vector control (V). After 48 hours, the cells were serum-starved for 2 hours and stimulated with HIV-1gp120 (500 ng/ml) or a control for 15 minutes as indicated. GAPDH used as loading control.</p

    Slit2N inhibits M-gp120-induced association of LSP1, WASp, Arp2/3, and β-actin in iMDDCs.

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    <p>(<b>A</b>) Representative immunoprecipitation of LSP1 with WASp, Arp2 and β-actin in iMDDCs incubated for various times with M-gp120. LSP1 used as loading control. (<b>B</b>) Quantitative analysis of the immunoprecipitation of LSP1 with WASp, Arp2 and β-actin in iMDDCs incubated for various times with M-gp120. LSP1 used as loading control. The band intensity in each lane was determined by densitometry. The fold change was determined by calculating the value of each lane vs. the unstimulated control (0 h, M-gp120 “−”). Data represent the mean ± SD of 3 independent experiments (*p≤0.05, **p≤0.01). (<b>C</b>) Representative immunoprecipitation of LSP1 with WASp, Arp2 and β-actin in untreated iMDDCs, and in iMDDCs incubated with M-gp120, Slit negative control then M-gp120, Slit2N then M-gp120, or Slit2N alone. LSP1 used as loading control. (<b>D</b>) Quantitative analysis of the immunoprecipitation of LSP1 with WASp, Arp2 and β-actin in untreated iMDDCs, and in iMDDCs incubated with M-gp120, Slit negative control then M-gp120, Slit2N then M-gp120, or Slit2N alone. LSP1 used as loading control. The band intensity in each lane was determined by densitometry. The fold change was determined by calculating the value of each lane vs. the unstimulated control. Data represent the mean ± SD of 3 independent experiments (*p≤0.05, **p≤0.01). (<b>E</b>) Representative Western blot analysis of LSP1, WASp, Arp2 and β-actin in untreated iMDDCs, and in iMDDCs incubated with M-gp120, Slit negative control then M-gp120, Slit2N then M-gp120, or Slit2N alone.</p

    Slit2N inhibits M-gp120-induced signaling through Src, and activation of Pyk2, CDC42, Rac1, and paxillin.

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    <p>(<b>A</b>) CDC42 and Rac1 activation assay: Lysates of untreated iMDDCs, and iMDDCs incubated with M-gp120, Slit2 negative control then M-gp120, Slit2N then M-gp120, or Slit2N alone were incubated with activated PAK1-conjugated agarose. Binding of activated CDC42 and Rac1 to PAK1 was visualized by Western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#s4" target="_blank">Materials and Methods</a>. PAK1 used as loading control. Representative assay is shown. (<b>B</b>) Quantitative analysis of the CDC42 and Rac1 activation by Western blot analysis. The band intensity in each lane was determined by densitometry. The fold change was determined by calculating the value of each lane vs. the unstimulated control or as indicated. Data represent the mean ± SD of 3 independent experiments (*p<0.05, **p≤0.01). (<b>C</b>) Representative Western blot analysis of total and phosphorylated Src, Pyk2 and paxillin in untreated iMDDCs, and in iMDDCs incubated with M-gp120, Slit2 negative control then M-gp120, Slit2N then M-gp120, or Slit2N alone. GAPDH used as loading control. (<b>D</b>) Quantitative analysis of phosphorylated Src, Pyk2 and paxillin by Western blot analysis in untreated iMDDCs, and in iMDDCs incubated with M-gp120, Slit2 negative control then M-gp120, Slit2N then M-gp120, or Slit2N alone. GAPDH used as loading control. The band intensity in each lane was determined by densitometry. The fold change was determined by calculating the value of each lane vs. the unstimulated control or as indicated. Data represent the mean ± SD of 3 independent experiments (*p<0.05, **p≤0.01).</p

    Slit2N inhibits HIV-1-gp120-induced migration and podosome formation of iMDDCs.

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    <p>(<b>A</b>) iMDDC transwell migration: cells were incubated with Slit2N, Slit2 negative control, or media alone for 2 hours, then seeded to the upper compartments of transwell chambers. Media +/− M-gp120 or Slit2N was added to the bottom compartments of the chambers. After 3 hours, iMDDCs that had migrated to the lower chambers were counted by hemocytometer. Data indicate the mean ± SD of 5 independent experiments (***p≤0.001). (<b>B</b>) iMDDC transendothelial migration: HUVECs were seeded into the upper compartment of transwell chambers and incubated at 37°C, 5% CO<sub>2</sub>, to confluency. iMDDCs were incubated with Slit2N, Slit2 negative control, or media alone for 2 hours, then seeded to the upper compartments of transwell chambers. Media +/− M-gp120 or Slit2N was added to the bottom compartments of the chambers. After 8 hours, iMDDCs that had migrated to the lower chambers were counted by hemocytometer. Data indicate the mean ± SD of 5 independent experiments (***p≤0.001). (<b>C</b>) Vinculin expression by confocal microscopy. iMDDCs were cultured on chamber slides and left untreated or incubated with M-gp120, Slit2N then M-gp120, or Slit2N alone (Slit2N incubation: 2 hours; M-gp120 incubation: 1 hour) before fixing and staining cells. Green  =  Vinculin; Blue  =  DAPI. Scale bars  = 2 µm. Arrows indicate a dense accumulation of podosomes at the cell's leading edge (“M-gp120” image), a reduced number of podosomes clustered at a weaker leading edge (“Slit2N + M-gp120” image), and a reduced number of podosomes localized around the cell's periphery (“Slit2N” image). Representative images are shown. (<b>D</b>) Percent of cell area covered by podosomes in the slides from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#pone-0048854-g001" target="_blank">Figure 1C</a>, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#s4" target="_blank">Materials and Methods</a>. (<b>E</b>) Number of podosomes per cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#s4" target="_blank">Materials and Methods</a>. Cells were treated as above. Data indicate the mean ± SD of 3 independent experiments x 50 cells per experimental condition (***p≤0.001).</p

    The differential effects of HIV-1 gp120 concentrations on Slit2 expression in L-LECs.

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    <p>Representative RT-PCR analysis (DNA gel) of Slit2 expression in L-LECs after incubation with designated concentrations of HIV-1 gp120 for 18 hours prior to performing RT-PCR. β-actin was amplified as an internal control.</p

    Slit2N inhibits M-gp120-induced colocalization of LSP1, WASp, Arp2/3, and β-actin to iMDDC podosomes.

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    <p>(<b>A</b>) Expression and colocalization of LSP1, WASp, Arp2/3 and β-actin by confocal microscopy. iMDDCs were cultured on chamber slides and left untreated or incubated with M-gp120, Slit2N then M-gp120, or Slit2N alone (Slit2N incubation: 2 hours; M-gp120 incubation: 1 hour) before fixing and staining cells. Green  =  LSP1; Red  =  WASp, Arp2/3, or β-actin, as indicated; Yellow/orange  =  merge; Blue  =  DAPI. In the “Merge” images: no arrow indicates a rounded cellular morphology, which is characteristic of non-migrating iMDDCs; one arrow indicates a single lamellipodium, densely populated with podosomes, at the leading edge of a polarized cell, which is characteristic of migrating iMDDCs; multiple arrows indicate multiple lamellipodia/focal adhesions with various orientations, which are characteristic of non-migrating iMDDCs. Scale bars  = 2 µm. Representative images are shown. (<b>B</b>) Quantitative analysis of the colocalization of LSP1 with (I) WASp, (II) Arp2/3 and (III) β-actin in iMDDCs, under conditions identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#pone-0048854-g004" target="_blank">Figure 4B</a>, using confocal microscopy and Volocity® software. Data represent the mean ± SD of 3 independent experiments x 3 randomly chosen cells per condition (*p≤0.05,**p≤0.01).</p

    Slit2N and Robo4 influence gp120-induced hyperpermeability in L-LECs by modulating c-Src kinase activation and signaling.

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    <p>(<b>A</b>) Representative Western blot analysis of phosphorylated c-Src in L-LECs after preincubation with either Slit2N (500 ng/ml) or a control for 2 hours before treatment with HIV-1 gp120 (500 ng/ml) for times indicated. GAPDH used as loading control. (<b>B</b>) Robo4 immunoprecipitation of Myc-tagged Slit2 by Western blot analysis in 293 cells. 293 s were co-transfected with a Robo4 expression plasmid and either a Myc-tagged Slit2 expression plasmid or a vector control. Cells were incubated for 48 hours before the protein from total cell lysates was collected for Robo4 immunoprecipitation. (<b>C</b>) Representative Western blot analysis of phosphorylated c-Src in L-LECs after transfection with either Robo4-specific siRNAs or a control siRNA for 48 hours before treatment with HIV-1 gp120 (500 ng/ml) for times indicated. GAPDH used as loading control. (<b>D</b>) Permeability through an L-LEC monolayer as previously described. An L-LEC monolayer was pretreated with a Src kinase inhibitor (2 µM) or DMSO for 2 hours before incubating with HIV-1 gp120 (500 ng/ml) or a control for 18 hours. Data indicate the mean ± SD of 3 independent experiments. (**p<0.01 for treatment with the Src kinase inhibitor versus DMSO control).</p

    Slit2N attenuates HIV-1 virus-induced lymphatic hyperpermeability.

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    <p>(<b>A</b>) Permeability through a dermal lymphatic endothelial cell (D-LEC) monolayer as previously described. D-LEC monolayers were pretreated with Slit2N (500 ng/ml) or a control for 2 hours before incubating with M-gp120 (500 ng/ml) for 18 hours. (<b>B</b>) Permeability through an L-LEC monolayer as previously described. L-LEC monolayers were pretreated with Slit2N (500 ng/ml) or a control for 1 hour, followed by incubation with HIV-1 virions (4.0×10<sup>6</sup> TCID 50/ml) or gp120 (500 ng/ml) for 5 hours. The viral load corresponds to a gp120 concentration of 425 ng/ml. For (<b>A</b>) and (<b>B</b>), data indicate the mean ± SD of 3 independent experiments. (*p<0.05; *** p<0.001 for treatment with Slit2N versus vehicle control).</p

    Hypothetical model of the effects of Slit2/Robo1 on HIV-1-gp120-induced podosome formation and migration of iMDDCs.

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    <p>(<b>A</b>) HIV-1-gp120 induces podosome formation, and the migration of iMDDCs. (<b>B</b>) Slit2/Robo1 inhibit HIV-1-gp120-induced podosome formation and migration of iMDDCs.</p
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