Journal: PLoS Pathogens

Article Title: Slit2/Robo4 Signaling Modulates HIV-1 gp120-Induced Lymphatic Hyperpermeability

doi: 10.1371/journal.ppat.1002461

Figure Lengend Snippet: ( A ) Representative Western blot analysis of fibronectin (FN) expression in L-LEC cytoplasm and supernatant. Cells were serum-starved for 1 hour and incubated with indicated concentrations of HIV-1 gp120 for 18 hours before harvesting protein. GAPDH used as loading control. ( B ) Robo4 and FN expression in L-LECs by confocal microscopy. L-LECs were cultured in chamber slides and incubated with either HIV-1 gp120 (500 ng/ml), Slit2 (500 ng/ml) or a control for 15 minutes before fixing and staining cells. Red = Robo4; Green = FN; Blue = DAPI. Scale bars = 10 µm. ( C ) Robo4 immunoprecipitation of total and phosphorylated FN (p-Ser/Ther) by Western blot analysis in L-LECs. Cells were incubated with either HIV-1 gp120 (500 ng/ml) or a control for 15 minutes before protein from total cell lysates was collected for Robo4 immunoprecipitation. Membrane was stripped and reprobed for Robo4 expression as a loading control.

Article Snippet: Specific Robo4 siRNAs and control siRNAs, purchased from Santa Cruz Biotechnology, Inc., were used to transfect L-LECs using HiPerFect transfection reagent from Qiagen, Inc.

Techniques: Western Blot, Expressing, Incubation, Control, Confocal Microscopy, Cell Culture, Staining, Immunoprecipitation, Membrane

Journal: PLoS Pathogens

Article Title: Slit2/Robo4 Signaling Modulates HIV-1 gp120-Induced Lymphatic Hyperpermeability

doi: 10.1371/journal.ppat.1002461

Figure Lengend Snippet: ( A ) Robo4 immunoprecipitation of α 5 β 1 integrin by Western blot analysis in L-LECs. Cells were incubated with either Slit2N (500 ng/ml), HIV-1 gp120 (500 ng/ml) or their respective controls (“−”) for 15 minutes before protein from total cell lysates was collected for Robo4 immunoprecipitation. Membranes were stripped and reprobed for Robo4 expression as a loading control. ( B ) Robo4 immunoprecipitation of α 5 β 1 integrin by Western blot analysis in L-LECs. Cells were incubated with either Slit2N (500 ng/ml) or a control for 2 hours and then treated with HIV-1 gp120 (500 ng/ml) for times indicated before the protein from total cell lysates was collected for Robo4 immunoprecipitation. Membrane was stripped and reprobed for Robo4 expression as a loading control. ( C ) Permeability through an L-LEC monolayer as previously described. L-LEC monolayers were pretreated with Slit2N (500 ng/ml) or a 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 for treatment with Slit2N versus vehicle control).

Article Snippet: Specific Robo4 siRNAs and control siRNAs, purchased from Santa Cruz Biotechnology, Inc., were used to transfect L-LECs using HiPerFect transfection reagent from Qiagen, Inc.

Techniques: Immunoprecipitation, Western Blot, Incubation, Expressing, Control, Membrane, Permeability

Journal: PLoS Pathogens

Article Title: Slit2/Robo4 Signaling Modulates HIV-1 gp120-Induced Lymphatic Hyperpermeability

doi: 10.1371/journal.ppat.1002461

Figure Lengend Snippet: ( A ) 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 ) 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. ( C ) 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. ( D ) 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).

Article Snippet: Specific Robo4 siRNAs and control siRNAs, purchased from Santa Cruz Biotechnology, Inc., were used to transfect L-LECs using HiPerFect transfection reagent from Qiagen, Inc.

Techniques: Western Blot, Control, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Incubation, Permeability

Journal: PLoS Pathogens

Article Title: Slit2/Robo4 Signaling Modulates HIV-1 gp120-Induced Lymphatic Hyperpermeability

doi: 10.1371/journal.ppat.1002461

Figure Lengend Snippet: ( A ) Representative Western blot analysis of Robo4 expression in L-LECs, 24 hours after transfection with a mixture of Robo4-specific siRNAs or a negative control siRNA. GAPDH used as loading control. ( B ) Permeability through an L-LEC monolayer as previously described. L-LECs transfected with control siRNAs or Robo4-specific siRNAs were seeded into the upper chamber of transwell plates and incubated with Slit2N (500 ng/ml), 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 Slit2N or HIV-1 gp120 versus negative control of the L-LECs transfected with control siRNAs).

Article Snippet: Specific Robo4 siRNAs and control siRNAs, purchased from Santa Cruz Biotechnology, Inc., were used to transfect L-LECs using HiPerFect transfection reagent from Qiagen, Inc.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Control, Permeability, Incubation

Journal: PLoS Pathogens

Article Title: Slit2/Robo4 Signaling Modulates HIV-1 gp120-Induced Lymphatic Hyperpermeability

doi: 10.1371/journal.ppat.1002461

Figure Lengend Snippet: ( A ) 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 ) 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). ( C ) 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.

Article Snippet: Specific Robo4 siRNAs and control siRNAs, purchased from Santa Cruz Biotechnology, Inc., were used to transfect L-LECs using HiPerFect transfection reagent from Qiagen, Inc.

Techniques: Western Blot, Control, Permeability, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Incubation

Journal: PLoS Pathogens

Article Title: Slit2/Robo4 Signaling Modulates HIV-1 gp120-Induced Lymphatic Hyperpermeability

doi: 10.1371/journal.ppat.1002461

Figure Lengend Snippet: Robo4 and α 5 β 1 integrin are expressed on the surface of lymphatic endothelial cells. Under physiologic conditions, endogenous Slit2 interacts with Robo4 via its Ig domains, and inhibits c-Src signaling. FN also interacts with Robo4 (via its FN type III domains), and binds to α 5 β 1 integrin on the endothelial cell surface. This contributes further to the dynamic regulation of Robo4 signaling by Slit2 and FN, to maintain the integrity of the lymphatic endothelial barrier. Upon HIV infection, gp120 significantly elevates FN levels and complexes with FN. FN then activates α 5 β 1 integrin, which results in enhanced intracellular signaling through α 5 β 1 integrin, a stronger interaction between FN and Robo4, and disruption of Robo4 signaling. These changes activate the c-Src signaling pathway and induce hyperpermeability of the lymphatic endothelial barrier. Exogenous Slit2 may protect the lymphatic channels from HIV-induced vasculopathy by interacting with Robo4 to restore Robo4 signaling, block the c-Src pathway, and inhibit HIV-induced lymphatic hyperpermeability.

Article Snippet: Specific Robo4 siRNAs and control siRNAs, purchased from Santa Cruz Biotechnology, Inc., were used to transfect L-LECs using HiPerFect transfection reagent from Qiagen, Inc.

Techniques: Infection, Disruption, Blocking Assay

Journal: PLOS ONE

Article Title: Carvedilol suppresses ryanodine receptor-dependent Ca 2+ bursts in human neurons bearing PSEN1 variants found in early onset Alzheimer’s disease

doi: 10.1371/journal.pone.0291887

Figure Lengend Snippet: ( A ) Xestospongin c (3 μM) did not affect the Ca 2+ -bursts in the PSEN1 A246E neuron (N = 20). ( B ) Dantrolene (30 μM) did not affect the Ca 2+ -bursts in the PSEN1 A246E neuron (N = 21). ( C ) Carvedilol (30 μM) terminated the Ca 2+ -bursts in the PSEN1 A246E neuron (N = 98). ( D ) VK-Ⅱ-86 (10 μM) terminated the Ca 2+ -bursts in the PSEN1 A246E neuron (N = 18).

Article Snippet: Cyclopiazonic acid (030–17171), dantrolene (359–44501) and xestospongin c (244–00721) were purchased from FUJIFILM Wako Chemicals (Osaka, Japan).

Techniques: