Journal: Scientific Reports

Article Title: Platelet activation determines the severity of thrombocytopenia in dengue infection

doi: 10.1038/srep41697

Figure Lengend Snippet: Whole blood of healthy individual was incubated with or without DENV2 (3.4 MOI) and perfused over HUVEC monolayer under shear of 2.5 dyne/cm 2 in presence of mepacrine (label platelets). (A) Platelet thrombus area and (B) platelet-VWF string number and (C) string length were measured. Data are the mean ± SEM from ten view-fields in three independent experiments. The thrombus area, and string number and length were increased in presence of DENV2, *p < 0.05. The string length (calculated as median with highest and lowest value) was increased in presence of DENV2, **p < 0.005. (D) The 40X images show the platelet thrombus (marked by dotted area) and platelet- VWF string (marked by arrow) in absence (D1) and presence (D2) DENV2.

Article Snippet: The whole blood collected from healthy individuals was perfused over the petri plate confluent with human umbilical vein endothelial cells (HUVEC) from ATCC, USA as mentioned in our recent work .

Techniques: Incubation, Shear

Journal: Blood

Article Title: The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner

doi: 10.1182/blood-2012-01-408096

Figure Lengend Snippet: PLD activity is required for Stx B-induced VWF secretion. Confluent HUVECs were incubated with 0.2 μg/mL Stx1B (A) or Stx2B (B) for the indicated time, and PLD activity in cell lysates was assayed. Values are normalized to untreated cells (0 minutes) and shown as the mean ± SE for 3 experiments. (C) Confluent HUVECs in 24-well dishes were stimulated for 30 minutes with 0.2 μg/mL Stx1B or Stx2B. Secreted VWF propeptide was quantified by ELISA for each condition. Values shown are the mean ± SE from 3 replicates. Mean VWF propeptide concentrations were 6 ng/mL, 11 ng/mL, and 15 ng/mL for control, Stx1B-, and Stx2B-treated cells, respectively. (D) Confluent HUVECs in 24-well dishes were pretreated for 30 minutes with or without 25mM n-butanol or t-butanol, then stimulated for 30 minutes with 0.2 μg/mL Stx1B. Cell surface-associated VWF was quantified by ELISA in triplicate for each condition. Values shown are the mean ± SE from 4 independent experiments. (E) Confluent HUVECs were perfused at 2.5 dyn/cm2 with 200 ng/mL Stx2B, with or without preincubation with n-butanol or t-butanol. VWF strings were counted in 10 fields, and values are shown as the mean ± SE, normalized to the Stx2B condition for each experiment. Experiments were conducted ≥ 3 times with similar results. For each panel, values for Stx1B or Stx2B are significantly different from control cells (P < .05). (D-E) Inhibition by n-butanol is significant (P < .01).

Article Snippet: Confluent HUVECs on glass-bottomed dishes (MatTek) were incubated with Stx1B or Stx2B for 5 or 15 minutes, washed 3 times with PBS, fixed with 1% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes.

Techniques: Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control, Inhibition

Journal: Blood

Article Title: The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner

doi: 10.1182/blood-2012-01-408096

Figure Lengend Snippet: PLD1 knockdown inhibits VWF secretion. (A) HUVECs were transfected with a plasmid that encodes shRNA that targets human PLD1 or firefly luciferase (shLUC). Cell lysate were prepared 72 hours after transfection and probed for PLD1. Membrane was stripped and reprobed for β-actin. (B-C) Seventy-two hours after transfection, HUVECs were incubated with medium that contained Stx1B (B) or Stx2B (C). VWF secretion and deposition on HUVEC surface was measured as described in “Methods.” Results from 3 independent experiments were averaged and plotted. Values were significantly different from the control (Mock) condition (P < .01).

Article Snippet: Confluent HUVECs on glass-bottomed dishes (MatTek) were incubated with Stx1B or Stx2B for 5 or 15 minutes, washed 3 times with PBS, fixed with 1% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes.

Techniques: Knockdown, Transfection, Plasmid Preparation, shRNA, Luciferase, Membrane, Incubation, Control

Journal: Blood

Article Title: The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner

doi: 10.1182/blood-2012-01-408096

Figure Lengend Snippet: Stx1B and Stx2B activate RhoA and increase endothelial monolayer permeability. (A) Confluent HUVECs were treated with Stx1B or Stx2B for 5 or 15 minutes. Cells were then fixed, permeabilized, and stained for actin (green) and vinculin (red) as a marker for focal adhesions. Bars, 10 μm. (B) Confluent HUVECs on transwell filters were treated with 200 ng/mL Stx1B or Stx2B for 15 minutes or 1 hour. Monolayer permeability was measured by the penetration of FITC-dextrin to the bottom part of the transwell chambers. Results are shown as the mean ± SE for 5 independent experiments. Stx2B increased trans-endothelial permeability more than did Stx1B at both time points (P < .05). (C) HUVECs were treated with Stx1B or Stx2B for 10 or 30 minutes before lysis buffer was added. Cell lysates were probed for total RhoA or GTP-bound RhoA. The experiment was performed 3 times with similar results.

Article Snippet: Confluent HUVECs on glass-bottomed dishes (MatTek) were incubated with Stx1B or Stx2B for 5 or 15 minutes, washed 3 times with PBS, fixed with 1% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes.

Techniques: Permeability, Staining, Marker, Lysis

Journal: Blood

Article Title: The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner

doi: 10.1182/blood-2012-01-408096

Figure Lengend Snippet: Differential effects of PKCα and Rho inhibitors on Stx1B- and Stx2B-induced VWF secretion and PLD activation. HUVECs were preincubated with buffer or ROCK inhibitor Y27632 (100μM), Rho family GTPase inhibitor exoenzyme C3 (1 μg/mL), or PKCα inhibitor Gö6976 (2μM) before stimulation with Stx1B (A) or Stx2B (B) for 10 minutes. PLD activity was quantified as described in “Methods.” Graphs represent the average results of 3-5 independent experi-ments. HUVECs were treated with inhibitors before stimulation with Stx1B (C) or Stx2B (D). Cell-surface VWF under each condition was quantified as described in “Methods.” Values for each condition were normalized to the amount of VWF secreted by unstimulated cells. Data reflect the average results of 4 independent experiments. Comparisons with the reference condition are significantly different as indicated: *P < .05; **P < .01.

Article Snippet: Confluent HUVECs on glass-bottomed dishes (MatTek) were incubated with Stx1B or Stx2B for 5 or 15 minutes, washed 3 times with PBS, fixed with 1% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes.

Techniques: Activation Assay, Activity Assay

Journal: Blood

Article Title: The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner

doi: 10.1182/blood-2012-01-408096

Figure Lengend Snippet: Effect of PKCα knockdown and dominant-negative RhoA on VWF secretion induced by Stx1B and Stx2B. (A) HUVECs were transfected with siRNA against PKCα or GFP (Ctrl). Whole-cell lysates were prepared 72 hours later, and PKCα level was assessed by Western blot analysis. The membrane was stripped and reprobed for β-actin. (B) HUVECs were transfected with siRNA against PKCα or GFP, and cell surface VWF was assayed by ELISA 72 hours later. Values for each condition were normalized to the amount of VWF secreted by unstimulated cells. The graph represents the results of 3 independent experiments. **P < .01. (C-D) HUVECs were transfected with cDNA encoding EGFP or EGFP-DN RhoA. Seventy-two hours later, confluent cells were stimulated for 1 hour with either Stx1B (C) or Stx2B (D), washed, and stained with fluorescent VWF antibody. Numbers of WPBs per cell (stimulated or unstimulated) were counted for 10-30 cells from multiple optical fields. Experiments were conducted 4 times with similar results. Pairwise comparisons with or without Stx1B or Stx2B are significantly different (P < .01) except for the dnRho conditions.

Article Snippet: Confluent HUVECs on glass-bottomed dishes (MatTek) were incubated with Stx1B or Stx2B for 5 or 15 minutes, washed 3 times with PBS, fixed with 1% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes.

Techniques: Knockdown, Dominant Negative Mutation, Transfection, Western Blot, Membrane, Enzyme-linked Immunosorbent Assay, Staining

Journal: Blood

Article Title: The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner

doi: 10.1182/blood-2012-01-408096

Figure Lengend Snippet: Arf6 is involved in StxB but not histamine-evoked WPB exocytosis. (A) HUVECs were treated with Stx1B or Stx2B for 5 minutes before lysis buffer was added. Cell lysates were directly probed for Arf6, or pull-down assay was conducted to concentrate GTP-bound Arf6. The experiment was performed 5 times with similar results. (B) HUVECs in 24-well dishes were transfected with wild-type or dominant-negative (T17N) Arf6, and cell-surface ELISA assays were conducted 72 hours later after StxB treatment. Values for each condition were normalized to the amount of VWF secreted by unstimulated cells. The experiment was repeated twice with similar results. (C-E) HUVECs were transfected with cDNA encoding EGFP, CFP-Arf6, or CFP-DN Arf6. Seventy-two hours later, confluent cells were stimulated for 1 hour with Stx1B (C), Stx2B (D), or histamine (E); washed; and stained with fluorescent VWF antibody. Numbers of WPBs per cell were counted for 10-30 cells from multiple optical fields. Experiments were performed 3 times with similar results. Pairwise comparisons with or without Stx1B or Stx2B are significantly different (P < .01), except for the dnArf6 and Arf6 conditions.

Article Snippet: Confluent HUVECs on glass-bottomed dishes (MatTek) were incubated with Stx1B or Stx2B for 5 or 15 minutes, washed 3 times with PBS, fixed with 1% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes.

Techniques: Lysis, Pull Down Assay, Transfection, Dominant Negative Mutation, Enzyme-linked Immunosorbent Assay, Staining