Figure 3.
MALT-1 regulates IL-1β–induced Rho activation, mobilization of P-selectin and VWF, and neutrophil adhesion to activated endothelial cells. (A) MALT1 silencing suppresses IL-1β–induced Rho activation in endothelial cells. HUVECs were transfected with 200 nM of scRNA or MALT1 siRNA. After 48 hours, the transfected cells were treated with IL-1β for indicated time periods. Rho activation was measured as GTP-bound Rho as described in the Methods section. The left panel shows a representative blot (t-Rho in the blot indicates total Rho). The right panel shows the quantification of Rho-GTP concentrations from densitometric analysis of signals of immunoblots. Rho-GTP concentrations measured in scRNA-transfected cells and not treated with IL-1β were taken as 1, and other values were shown relative to this value. (B) MALT1 silencing inhibits IL-1β–induced translocation of P-selectin to the cell surface and secretion of VWF. HUVECs transfected with scRNA or MALT1 siRNA were treated with a control vehicle or IL-1β for 1 hour. After 1 hour, the cell supernatants were collected, and cell lysates were harvested. Cell supernatants were precipitated with TCA to concentrate proteins. Cell lysates were probed for MALT1, P-selectin, and VWF by immunoblot analysis; cell supernatants were probed for VWF by immunoblot analysis to assess the concentrations of secreted (S) VWF (left panel). The translocation of P-selectin to the cell surface was measured by cell surface ELISA (middle panel). VWF secretion was quantified by densitometric analysis of VWF (S) immunoblots (right panel). Concentrations of P-selectin on the cell surface and secreted VWF measured in cells transfected with scRNA and treated with a control vehicle were taken as 1.0, and other values were shown relative to these. (C) Pharmacological inhibition of MALT1 attenuates IL-1β–induced P-selectin translocation to the cell surface and secretion of VWF. HUVECs were treated with MALT1 inhibitor, mepazine (20 µM), or vehicle (veh) for 1 hour. After that, the cells were treated with a control vehicle (CV) or IL-1β for 1 hour. The assessment of P-selectin translocation to the cell surface and VWF secretion was performed as described in (B). (D-E) (D) MALT1 silencing or (E) pharmacological inhibition attenuates neutrophil adhesion to IL-1β–activated endothelial cells. HUVECs cultured on cover glasses were transfected with scRNA, MALT1 siRNA, or treated with MALT1 inhibitor, mepazine, as described in the figure legends of (B-C). HUVECs were stimulated with IL-1β for 1 or 6 hours. After washing the cells, PKH-labeled human neutrophils (5 × 105/mL) were added to endothelial cells. After 30 minutes, the unbound neutrophils were removed, and endothelial cells were washed. Neutrophils adhered to endothelial cells were fixed in 4% PFA for 15 minutes. The cells were visualized and imaged under 20× magnification using a confocal microscope (left panel). The cell count enumerated at 3 random locations of each cover glass of 3 independent experiments was shown in the bar graph (right panel). **P < .01 and ***P < .001.

MALT-1 regulates IL-1β–induced Rho activation, mobilization of P-selectin and VWF, and neutrophil adhesion to activated endothelial cells. (A) MALT1 silencing suppresses IL-1β–induced Rho activation in endothelial cells. HUVECs were transfected with 200 nM of scRNA or MALT1 siRNA. After 48 hours, the transfected cells were treated with IL-1β for indicated time periods. Rho activation was measured as GTP-bound Rho as described in the Methods section. The left panel shows a representative blot (t-Rho in the blot indicates total Rho). The right panel shows the quantification of Rho-GTP concentrations from densitometric analysis of signals of immunoblots. Rho-GTP concentrations measured in scRNA-transfected cells and not treated with IL-1β were taken as 1, and other values were shown relative to this value. (B) MALT1 silencing inhibits IL-1β–induced translocation of P-selectin to the cell surface and secretion of VWF. HUVECs transfected with scRNA or MALT1 siRNA were treated with a control vehicle or IL-1β for 1 hour. After 1 hour, the cell supernatants were collected, and cell lysates were harvested. Cell supernatants were precipitated with TCA to concentrate proteins. Cell lysates were probed for MALT1, P-selectin, and VWF by immunoblot analysis; cell supernatants were probed for VWF by immunoblot analysis to assess the concentrations of secreted (S) VWF (left panel). The translocation of P-selectin to the cell surface was measured by cell surface ELISA (middle panel). VWF secretion was quantified by densitometric analysis of VWF (S) immunoblots (right panel). Concentrations of P-selectin on the cell surface and secreted VWF measured in cells transfected with scRNA and treated with a control vehicle were taken as 1.0, and other values were shown relative to these. (C) Pharmacological inhibition of MALT1 attenuates IL-1β–induced P-selectin translocation to the cell surface and secretion of VWF. HUVECs were treated with MALT1 inhibitor, mepazine (20 µM), or vehicle (veh) for 1 hour. After that, the cells were treated with a control vehicle (CV) or IL-1β for 1 hour. The assessment of P-selectin translocation to the cell surface and VWF secretion was performed as described in (B). (D-E) (D) MALT1 silencing or (E) pharmacological inhibition attenuates neutrophil adhesion to IL-1β–activated endothelial cells. HUVECs cultured on cover glasses were transfected with scRNA, MALT1 siRNA, or treated with MALT1 inhibitor, mepazine, as described in the figure legends of (B-C). HUVECs were stimulated with IL-1β for 1 or 6 hours. After washing the cells, PKH-labeled human neutrophils (5 × 105/mL) were added to endothelial cells. After 30 minutes, the unbound neutrophils were removed, and endothelial cells were washed. Neutrophils adhered to endothelial cells were fixed in 4% PFA for 15 minutes. The cells were visualized and imaged under 20× magnification using a confocal microscope (left panel). The cell count enumerated at 3 random locations of each cover glass of 3 independent experiments was shown in the bar graph (right panel). **P < .01 and ***P < .001.

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