Journal: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: A. Western blots of phosphorylated Ets1 (pEts1), Ets1 and total Ras proteins in immortalized mouse LECs (mLECs) transfected with control siRNA or mixed siRNAs for ras genes ( Hras , Nras and Kras ) in the presence or absence of VEGF-A, VEGF-C or VEGF-D. B. Real-time RT-PCR assay for mRNAs in mLECs transfected with control, Ets1 and Ets2 siRNAs. si-1 and -2 represent two individual siRNAs. Ets1/2 si represents transfection with mixed siRNAs for Ets1 and Ets2. Error bars represent the S.D.; n = 3. *p<0.05, ***p<0.005, ****p<0.001 (vs. mLECs transfected with control siRNA; see and ). C. Protein expression in mLECs transfected with control, Ets1 and Ets2 siRNAs. Left panel, western blots; right panel, quantitative analysis of western blots. Error bars represent the S.D.; n = 3. **p<0.01, ***p<0.005, ****p<0.001 (vs. mLECs transfected with control siRNA).

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: A. VEGFR-3 and Gapdh protein expression in Vegfr3-knockdown LECs. Upper panel, western blots; lower panel, quantitative analysis of western blots. Error bars represent the S.D.; n = 3. ****p<0.001 (vs. mLECs transfected with control siRNA). B. WST-1 assays using Vegfr3-knockdown mLECs (upper left panel) and Ets-knockdown mLECs (lower left panel), and BrdU assays using Vegfr3-knockdown mLECs (upper right panel) and Ets-knockdown mLECs (lower right panel). Error bars represent the S.D.; n = 12. *p<0.05, ****p<0.001 (vs. mLECs transfected with control siRNA in each assay). C. DiI-stained cellular networks of mLECs transfected with control, Ets1, Ets2, and Vegfr3 siRNAs on Matrigel. Scale bar = 500 µm. DiI-labeled areas were quantified and the mean area of DiI-labeled wild-type mLECs was normalized to 1. Error bars represent the S.D.; n = 3. **p<0.01, ***p<0.005, ****p<0.001 (vs. mLECs transfected with control siRNA).

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Expressing, Knockdown, Western Blot, Transfection, Control, Staining, Labeling

Journal: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: A. Real-time RT-PCR assay for VEGFR3 mRNA in primary human LECs (hLECs) transfected with control and ETS1 siRNAs. si-1 and -2 represent two individual siRNAs. Error bars represent the S.D.; n = 3. ***p<0.005, ****p<0.001 (vs. hLECs transfected with control siRNA; see ). B. WST-1 assays and BrdU assays using ETS1-knockdown hLECs. Error bars represent the S.D.; n = 12. ***p<0.005, ****p<0.001 (vs. hLECs transfected with control siRNA). C. DiI-stained cellular networks of hLECs transfected with control and ETS1 siRNAs on Matrigel. Scale bar = 500 µm. DiI-labeled areas were quantified and the mean area of DiI-labeled wild-type hLECs was normalized to 1. Error bars represent the S.D.; n = 3. *p<0.05, ****p<0.001 (vs. hLECs transfected with control siRNA).

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Transfection, Control, Knockdown, Staining, Labeling

Journal: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: The Ras/MAPK/Ets pathway involved in transcriptional regulation of the Vegfr3 gene in LECs. Ras/MAPK signal-activated Ets proteins, together with p300, regulate Vegfr3 gene expression and lead to changes in cellular proliferation and morphogenesis.

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Gene Expression

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 ) Permeability, as determined by the translocation of FITC-conjugated dextran particles through an L-LEC monolayer in transwell chambers. Cells were incubated with M-gp120, T-gp120 or controls at various concentrations for 18 hours. Dextran particles were added, and fluorescence assessed after 5 minutes. Permeability was calculated based on the relative fluorescence of media in lower chambers of HIV-1 gp120-treated cells vs. the controls (“0”). Data indicate the mean ± SD of 3 independent experiments. (*** p<0.001). ( B ) Immunofluorescent analysis of CXCR4 expression in L-LECs. Scale bars = 20 µm. Representative images are shown.

Article Snippet: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, Inc.).

Techniques: Permeability, Translocation Assay, Incubation, Fluorescence, Expressing

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: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, 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: 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.

Article Snippet: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Amplification, Control

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 α 5 β 1 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 ) Phosphorylated α 5 β 1 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. ( C ) Permeability through an L-LEC monolayer as previously described. L-LEC monolayers were pretreated with a neutralizing anti-integrin β 1 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).

Article Snippet: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, Inc.).

Techniques: Western Blot, Incubation, Control, Expressing, Confocal Microscopy, Cell Culture, Staining, 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 ) 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: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, 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: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, 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: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, 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: Primary human lung lymphatic endothelial cells (L-LECs) and dermal lymphatic endothelial cells (D-LECs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and maintained in EBM-2 medium with EGM-2MV SingleQuots (Lonza, Inc.).

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

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) A schematic of an organotypic 3D lymphatic vessel model (LV-on-chip). Prox-1 (green) and CD31 (red) expression confirms lymphatic endothelial identity and cell morphology in the channel. (B) Morphologic changes in human dermal microvascular blood endothelial cells (BECs) with lymphatic endothelial cells (LECs) after one day of cell seeding. BECs become more contractile than LECs, forming a smaller vessel diameter compared to LECs. (C) BVs and LVs observed in mouse ear tissues. mLYVE-1, anti-mouse LYVE-1 antibody; mCD31, anti-mouse CD31 antibody. (D) Phalloidin (red) and anti-VE-cad (VE-cadherin) antibody (green) staining to visualize F-actin and adherens junctions. (E) Lymphatic and blood vessel barrier function. 70 kDa dextran was introduced into the vessel lumens and dextran diffusion was observed in real time under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (F) Quantification of the permeability of BEC-generated engineered BVs and LEC-generated LVs. ** p = 0.0016, two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Expressing, Staining, Diffusion-based Assay, Microscopy, Permeability, Generated, Two Tailed Test

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) Lymphatic endothelial cells (LECs) in different ECM hydrogels (2D): 2.5 mg/ml collagen 1, 2.5 mg/ml collagen 1 and 150 μg/ml Fibronectin, and no gel (plastic). F-actin and VE-cad were visualized to assess cytoskeletal arrangement and adherens junction formation in each condition. (B) Quantification of the relative junction area was performed, illustrating a significantly lower junction area in cells grown on the 2.5 mg/ml collagen 1 compared to the cells grown directly on plastic. ** p = 0.0017 (Collagen 1 vs. plastic); higher junction area in cells grown on the 2.5 mg/ml collagen 1 + fibronectin compared to the cells grown on collagen 1. * p = 0.0151 (Collagen 1 + fibronectin vs. Collagen 1); not-significant (ns) p = 0.5292 (Collagen 1 + fibronectin vs plastic). One-way ANOVA with Tukey’s HSD tests , n = 6 per group. Data are expressed as mean ± S.E.M. (C) Dynamics of fibronectin on LECs in collagen 1 or collagen 1 + fibronectin gel. On collagen 1 gel, LEC islands with VE-cad expression lacks fibronectin expression. On collagen 1 + fibronectin, fibronectin connects separate LEC islands. (D) At day 4 on Collagen 1 + fibronectin, LECs showed tightened junctions and fibronectin was localized in the junctional area.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Expressing

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) Activated integrin α5 was visualized in both ECM composition conditions by using anti-integrin α5 antibody (clone: SNAKA51) that can only detect the activated form of the integrin α5. F-actin was also observed in these conditions. (B) LECs in Collagen 1 were pre-treated with anti-integrin α5 antibodies (clone: SNAKA51) antibodies to activate integrin α5 in LECs. The fixed samples were stained with anti-VE-cadherin antibodies, anti-JAM-A antibodies, and phalloidin to visualize adherens junctions and F-actin. (C) Quantification of the relative junction area was performed, illustrating a significantly higher junction area in integrin α5 activated cells compared to the control LECs. ** p = 0.0020; Two tailed unpaired Student t-test, n = 6 per group. Data are expressed as mean ± S.E.M. (D) Control LECs or LECs with activated integrin α5 were seeded in LV-on-chip and cultured for 3 days on the rocking platform. 70 kDa dextran was introduced to the lymphatic lumens. Dextran diffusion was observed at 0 and 1 minutes under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (E) Quantification of the permeability of LEC-generated engineered LVs in collagen 1 with and without integrin α5 activation. ** p = 0.0021. Two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M. (F) This table summarizes our findings regarding LEC permeability and integrin α5 activity. LVs grown in Collagen 1 without any activator treatment showed high LEC permeability and low integrin α5 activity. In contrast, LVs grown in either Collagen 1 + Fibronectin or LVs grown in only Collagen 1 with integrin α5 activator pre-treatment both showed low LEC permeability and high integrin α5 activity.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Staining, Control, Two Tailed Test, Cell Culture, Diffusion-based Assay, Microscopy, Permeability, Generated, Activation Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

doi: 10.1016/j.jbc.2021.100299

Figure Lengend Snippet: Quantitative analysis of A4GALT transcripts in CHO-Lec2 cells

Article Snippet: CHO-Lec2 cells were obtained from the American Type Culture Collection.

Techniques: