Figure 1.
Gab2 is essential for IL-1β–induced exocytosis of P-selectin and VWF in endothelial cells and the adhesion of neutrophils to activated endothelial cells. (A) IL-1β induces P-selectin expression on the endothelial cell surface. HUVECs were treated with IL-1β (10 ng/mL) or TNFα (10 ng/mL) alone or in combination for 1 hour. At the end of 1 hour, the cells were fixed in 2% paraformaldehyde (PFA), and the surface expression of P-selectin was measured by cell surface ELISA. Since the inclusion of TNFα in IL-1β treatment gave a robust increase in P-selectin translocation to the cell surface, in all experiments described in this and other figures that examined the exocytosis of P-selectin and VWF, IL-1β was supplemented with TNFα in treating endothelial cells. (B) Gab2 silencing does not affect total P-selectin concentrations. HUVECs were transfected with 200 nM scrambled RNA (scRNA) or Gab2 small interfering RNA(siRNA). After 48 hours, the cells were treated with IL-1β for 1 hour. The cells were lysed, and the concentrations of Gab2 and P-selectin were analyzed by immunoblotting. Densitometric analysis of immunoblots showed that Gab2 silencing knocked down around 85% of Gab2 and no change in the total P-selectin. (C) Gab2 silencing impairs the translocation of P-selectin to the cell surface. HUVECs transfected with scRNA or Gab2 siRNA were treated with IL-1β for 1 hour. The cell surface expression of P-selectin was measured by cell surface ELISA. (D-E) Gab2 silencing impairs IL-1β–induced expression of P-selectin at the cell surface without impairing the total cellular expression of P-selectin. HUVECs, cultured on cover glasses, were transfected with 200 nM of scRNA or Gab2 siRNA. After 48 hours, the cells were treated with IL-1β for 1 hour. P-selectin expression was analyzed in (D) unpermeabilized or (E) permeabilized cells by immunofluorescence microscopy. (F) Gab2 silencing suppresses IL-1β–induced secretion of VWF. HUVECs transfected with scRNA or Gab2 siRNA were treated with IL-1β for 1 hour. At the end of 1 hour, cell supernatants were removed and precipitated with trichloroacetic acid (TCA) (6%) to concentrate proteins. The suspension of TCA precipitates was subjected to immunoblot analysis to measure secreted (S) VWF. As a control, VWF in cell lysates was also analyzed by immunoblot analysis. Concentrations of secreted VWF were quantified by densitometric analysis of VWF (S) band, and the value obtained in control scRNA transfected cells was arbitrarily assigned as 1. The top panel shows representative immunoblot, and the bottom panel shows densitometric quantification of the VWF (S) band. (G) P-selectin-dependent adhesion of neutrophils to activated endothelial cells. HUVECs cultured on cover glasses were treated with IL-1β for 1 or 6 hours. The cells were washed and incubated with a P-selectin–blocking monoclonal antibody or control IgG (10 µg/mL) for 1 hour. After that, monolayers of HUVECs were layered with PKH-labeled human neutrophils (5 × 105/mL). Neutrophils were allowed to adhere for 30 minutes. Then, unbound neutrophils were removed, the endothelial cell monolayers were washed, and the adhered neutrophils to endothelial cells were fixed in 4% PFA for 15 minutes. The cover glass was mounted with antifade Fluro gel, and the cells were visualized under a confocal microscope at 20× magnification. The micrographs shown were the representative images of 3 independent experiments. The adhered cells were enumerated at 3 random locations in each cover glass, and these data were presented in the right-side panel. (H) Gab2 silencing in endothelial cells attenuates neutrophil adhesion to activated endothelial cells. HUVECs transfected with scRNA or Gab2 siRNA were treated with IL-1β, and the adhesion of neutrophils to activated endothelial cells was analyzed as described in panel (G). (I) IL-1β–induced expression of VCAM1. HUVECs were treated with IL-1β for 1 or 6 hours. The cell lysates were probed for VCAM1 by immunoblot analysis. Data are the mean ± standard deviation of 3 independent experiments. *P < .05, **P < .01, and ***P < .001. ns, no statistically significant difference.

Gab2 is essential for IL-1β–induced exocytosis of P-selectin and VWF in endothelial cells and the adhesion of neutrophils to activated endothelial cells. (A) IL-1β induces P-selectin expression on the endothelial cell surface. HUVECs were treated with IL-1β (10 ng/mL) or TNFα (10 ng/mL) alone or in combination for 1 hour. At the end of 1 hour, the cells were fixed in 2% paraformaldehyde (PFA), and the surface expression of P-selectin was measured by cell surface ELISA. Since the inclusion of TNFα in IL-1β treatment gave a robust increase in P-selectin translocation to the cell surface, in all experiments described in this and other figures that examined the exocytosis of P-selectin and VWF, IL-1β was supplemented with TNFα in treating endothelial cells. (B) Gab2 silencing does not affect total P-selectin concentrations. HUVECs were transfected with 200 nM scrambled RNA (scRNA) or Gab2 small interfering RNA(siRNA). After 48 hours, the cells were treated with IL-1β for 1 hour. The cells were lysed, and the concentrations of Gab2 and P-selectin were analyzed by immunoblotting. Densitometric analysis of immunoblots showed that Gab2 silencing knocked down around 85% of Gab2 and no change in the total P-selectin. (C) Gab2 silencing impairs the translocation of P-selectin to the cell surface. HUVECs transfected with scRNA or Gab2 siRNA were treated with IL-1β for 1 hour. The cell surface expression of P-selectin was measured by cell surface ELISA. (D-E) Gab2 silencing impairs IL-1β–induced expression of P-selectin at the cell surface without impairing the total cellular expression of P-selectin. HUVECs, cultured on cover glasses, were transfected with 200 nM of scRNA or Gab2 siRNA. After 48 hours, the cells were treated with IL-1β for 1 hour. P-selectin expression was analyzed in (D) unpermeabilized or (E) permeabilized cells by immunofluorescence microscopy. (F) Gab2 silencing suppresses IL-1β–induced secretion of VWF. HUVECs transfected with scRNA or Gab2 siRNA were treated with IL-1β for 1 hour. At the end of 1 hour, cell supernatants were removed and precipitated with trichloroacetic acid (TCA) (6%) to concentrate proteins. The suspension of TCA precipitates was subjected to immunoblot analysis to measure secreted (S) VWF. As a control, VWF in cell lysates was also analyzed by immunoblot analysis. Concentrations of secreted VWF were quantified by densitometric analysis of VWF (S) band, and the value obtained in control scRNA transfected cells was arbitrarily assigned as 1. The top panel shows representative immunoblot, and the bottom panel shows densitometric quantification of the VWF (S) band. (G) P-selectin-dependent adhesion of neutrophils to activated endothelial cells. HUVECs cultured on cover glasses were treated with IL-1β for 1 or 6 hours. The cells were washed and incubated with a P-selectin–blocking monoclonal antibody or control IgG (10 µg/mL) for 1 hour. After that, monolayers of HUVECs were layered with PKH-labeled human neutrophils (5 × 105/mL). Neutrophils were allowed to adhere for 30 minutes. Then, unbound neutrophils were removed, the endothelial cell monolayers were washed, and the adhered neutrophils to endothelial cells were fixed in 4% PFA for 15 minutes. The cover glass was mounted with antifade Fluro gel, and the cells were visualized under a confocal microscope at 20× magnification. The micrographs shown were the representative images of 3 independent experiments. The adhered cells were enumerated at 3 random locations in each cover glass, and these data were presented in the right-side panel. (H) Gab2 silencing in endothelial cells attenuates neutrophil adhesion to activated endothelial cells. HUVECs transfected with scRNA or Gab2 siRNA were treated with IL-1β, and the adhesion of neutrophils to activated endothelial cells was analyzed as described in panel (G). (I) IL-1β–induced expression of VCAM1. HUVECs were treated with IL-1β for 1 or 6 hours. The cell lysates were probed for VCAM1 by immunoblot analysis. Data are the mean ± standard deviation of 3 independent experiments. *P < .05, **P < .01, and ***P < .001. ns, no statistically significant difference.

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