American Society of Hematology

Figure 2

$Figure 2. Cellular PLD activity and plasma membrane PA levels increase upon stimulation of endothelial secretion. (A) PLD activity in resting and histamine-stimulated HUVECs. After a 10-minute treatment with histamine-free or histamine-containing medium, cells were lysed and assayed for PLD activity. The effect on PLD activity was quantified in sets of independent experiments (number of experiments shown at the base of each column) and statistical significance (*P < .01) was calculated by unpaired Student t test. Bars represent mean plus or minus SEM. (B) PA generation at the plasma membrane (PM) in response to histamine-stimulation. HUVECs were transfected with the PABD of Raf1 fused to EGFP (PABD-GFP). Six hours after transfection, cells were incubated for 10 minutes with basal medium either without (top panels) or with (bottom panels) 100 μM histamine. Stimulation of exocytosis triggered the recruitment of a fraction of the PA sensor to the plasma membrane as visualized by costaining with syntaxin 4. In the mask images, only the double-labeled pixels are displayed, visualizing areas of colocalization of PABD-GFP with syntaxin 4. Bars represent 10 μm. (C) The fluorescence intensity of GFP-PABD at the PM relative to that in the cytosol was determined in 5 representative cells from independent experiments using the Zeiss LSM instrument software 3.2. Average fluorescence intensities were determined by randomly choosing areas of defined size covering the plasma membrane and a cytosolic region of identical size 5 μm away from the plasma membrane. Ratios of plasma membrane to cytosolic pixel fluorescence intensity were then calculated, and statistical significance (*P < .01) of the results was evaluated by unpaired Student t tests. Bars represent mean plus or minus the standard error of the mean. (D) Stimulation with histamine triggers a recruitment of PLD1 to the plasma membrane. HUVECs were transiently transfected with a PLD1-GFP expression plasmid. Six hours after transfection, cells were stimulated by addition of 100 μM histamine to the culture medium. Confocal images show a live cell before (resting) and 5 minutes after addition of histamine (stimulated). Note the plasma membrane localization in stimulated cells (arrowheads). Bar represents 10 μm. (E) The fluorescence intensity of GFP-PLD1 at the PM relative to that in the cytosol at 5 minutes after histamine stimulation was determined in 10 representative cells from independent experiments using the Zeiss LSM instrument software 3.2. Bars represent mean plus or minus SEM, and statistical significance (*P < .01) was calculated by unpaired Student t test.$

Cellular PLD activity and plasma membrane PA levels increase upon stimulation of endothelial secretion. (A) PLD activity in resting and histamine-stimulated HUVECs. After a 10-minute treatment with histamine-free or histamine-containing medium, cells were lysed and assayed for PLD activity. The effect on PLD activity was quantified in sets of independent experiments (number of experiments shown at the base of each column) and statistical significance (*P < .01) was calculated by unpaired Student t test. Bars represent mean plus or minus SEM. (B) PA generation at the plasma membrane (PM) in response to histamine-stimulation. HUVECs were transfected with the PABD of Raf1 fused to EGFP (PABD-GFP). Six hours after transfection, cells were incubated for 10 minutes with basal medium either without (top panels) or with (bottom panels) 100 μM histamine. Stimulation of exocytosis triggered the recruitment of a fraction of the PA sensor to the plasma membrane as visualized by costaining with syntaxin 4. In the mask images, only the double-labeled pixels are displayed, visualizing areas of colocalization of PABD-GFP with syntaxin 4. Bars represent 10 μm. (C) The fluorescence intensity of GFP-PABD at the PM relative to that in the cytosol was determined in 5 representative cells from independent experiments using the Zeiss LSM instrument software 3.2. Average fluorescence intensities were determined by randomly choosing areas of defined size covering the plasma membrane and a cytosolic region of identical size 5 μm away from the plasma membrane. Ratios of plasma membrane to cytosolic pixel fluorescence intensity were then calculated, and statistical significance (*P < .01) of the results was evaluated by unpaired Student t tests. Bars represent mean plus or minus the standard error of the mean. (D) Stimulation with histamine triggers a recruitment of PLD1 to the plasma membrane. HUVECs were transiently transfected with a PLD1-GFP expression plasmid. Six hours after transfection, cells were stimulated by addition of 100 μM histamine to the culture medium. Confocal images show a live cell before (resting) and 5 minutes after addition of histamine (stimulated). Note the plasma membrane localization in stimulated cells (arrowheads). Bar represents 10 μm. (E) The fluorescence intensity of GFP-PLD1 at the PM relative to that in the cytosol at 5 minutes after histamine stimulation was determined in 10 representative cells from independent experiments using the Zeiss LSM instrument software 3.2. Bars represent mean plus or minus SEM, and statistical significance (*P < .01) was calculated by unpaired Student t test.

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