19 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 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 enhances Robo1, WASp, and LSP1 association, and their colocalization around the periphery of iMDDCs.

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    <p>(<b>A</b>) Representative immunoprecipitation of Robo1 with WASp, LSP1, 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. Robo1 used as loading control. (<b>B</b>) Quantitative analysis of the immunoprecipitation of Robo1 with WASp, LSP1, 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. Robo1 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>C</b>) Representative Western blot analysis of Robo1 expression in untreated iMDDCs, and in iMDDCs incubated with M-gp120, Slit2 negative control then M-gp120, Slit2N then M-gp120, or Slit2N alone. β-actin used as loading control. (<b>D</b>) Expression and colocalization of Robo1, WASp, and LSP1 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. Red  =  Robo1; Green  =  WASp or LSP1, as indicated; Yellow/orange  =  merge; Blue  =  DAPI. In the “Merge” images: no arrow indicates a rounded cellular morphology with diffuse or no colocalization of the proteins; one arrow indicates a single lamellipodium, but little or no colocalization of proteins to the podosomes at the leading edge of the iMDDC; multiple arrows indicate colocalization of proteins around the periphery of the cells, or in multiple lamellipodia with various orientations. Scale bars  = 2 µm. Representative images are shown. (<b>E</b>) Quantitative analysis of the colocalization of Robo1 with (I) WASp and (II) LSP1 in iMDDCs, under conditions identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#pone-0048854-g005" target="_blank">Figure 5D</a>, using confocal microscopy and Volocity® software. Data represent the mean ± SD of 3 independent experiments x 3 randomly chosen cells per condition (**p≤0.01, ***p≤0.001 for cells treated with Slit2N, then M-gp120 or just Slit2N vs. untreated control).</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 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

    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

    LSP1 contributes to M-gp120-induced migration and podosome formation of iMDDCs.

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    <p>(<b>A</b>) LSP1 expression by confocal microscopy. iMDDCs were cultured on chamber slides and left untreated or incubated with M-gp120 for 1 hour before fixing and staining cells. Red  =  LSP1; Blue  =  DAPI. Scale bars  = 2 µm. Arrow indicates the cell's leading edge. Representative images are shown. (<b>B</b>) Representative Western blot analysis of LSP1 expression in untreated iMDDCs and in iMDDCs transfected with NT-siRNAs or LSP1-specific siRNAs. β-actin used as loading control. (<b>C</b>) Quantitative analysis of LSP1 expression in untreated iMDDCs and in iMDDCs transfected with NT-siRNAs or LSP1-specific siRNAs. The band intensity in each lane of 3 independent Western blots (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048854#pone-0048854-g002" target="_blank">Figure 2B</a>) was determined by densitometry. Percent expression was calculated relative to the untreated control cells, whose LSP1 levels were established as 100%. Data represent the mean ± SD of 3 independent experiments (**p≤0.01). (<b>D</b>) LSP1 and vinculin expression by confocal microscopy. iMDDCs transfected with NT-siRNAs or with LSP1-specific siRNAs were cultured on chamber slides and incubated with M-gp120 for 1 hour before fixing and staining cells. Green  =  Vinculin; Red  =  LSP1; Blue  =  DAPI. Scale bars  = 2 µm. Arrows in the upper left panel and lower left panel indicate a dense accumulation of podosomes at the cells' leading edge; while arrows in the upper right panel and lower right panel indicate fewer podosomes, more diffusely distributed throughout cells without a distinct leading edge. Represent­ative images are shown. (<b>E</b>) Transwell Migration: iMDDCs and iMDDCs transfected with NT-siRNAs or with LSP1-specific siRNAs were seeded to the upper compartments of transwell chambers. Media +/− M-gp120 was added to the bottom compartments of the chambers. After 2 hours, cells that had migrated to the lower chambers were counted by hemocytometer. Data indicate the mean ± SD of 4 independent experiments (**p≤0.01).</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
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