Depletion of endothelial PLD1, but not PLD2, interferes with histamine-evoked secretion of VWF. (A) Quantification of VWF secretion from histamine-stimulated HUVECs depleted for PLD1 or PLD2. Forty-eight hours after transfection with shRNA plasmids specific for PLD1 or PLD2 or a nontargeting shRNA control, HUVECs were subjected to histamine treatment for 20 minutes. The amount of VWF released into the cell culture supernatant was then determined as described in “Methods.” The effect on secretion was quantified in sets of 9 independent experiments, and 1-way ANOVA with unpaired Student t test was performed to evaluate statistical significance (*P < .01). Bars represent mean plus or minus SEM. (B,C) Expression of a shRNA-insensitive PLD1 construct restores secretory responsiveness. Twenty-four hours after transfection with shRNA plasmids targeted at PLD1, cells were transfected with a pCGN-PLD1 rescue plasmid expressing a wobble position-mutated HA-tagged PLD1 cDNA insensitive to PLD1 shRNA. Twenty-four hours after the second transfection, HUVECs were used in immunofluorescence experiments or stimulated for secretion. (B) HA-tagged PLD1 was labeled using mouse monoclonal anti-HA antibodies, and cells expressing the shRNA constructs were identified by expression of plasmid-encoded dsRed. Note that the PLD1 rescue construct is stable in the shRNA-expressing cell. Bar represents 10 μm. (C) Cells transfected with shRNA and rescue plasmids were subjected to histamine treatment, and the amount of VWF released into the cell culture supernatant was determined as described in “Methods.” The effect on secretion was quantified in sets of 4 independent experiments, and 1-way ANOVA with unpaired Student t test was performed to evaluate statistical significance (*P < .01). Bars represent mean plus or minus SEM. Note that histamine-evoked secretion of VWF is restored by coexpression of the pCGN-PLD1 rescue plasmid.