KLF2 increases VWF secretion in HUVEC cultures by inducing a homogenous distribution of WPBs over the cell population. (A) Histogram of the number of WPBs that were counted in randomly chosen cells in mock (□) and KLF2 (▪) lentivirus-transduced HUVECs. Prolonged KLF2 expression induces a more homogeneous Gaussian distribution of the WPB number per cell over the entire cell population. (B) The thrombin- and forskolin-stimulated secretion of VWF in the medium was assayed after 0 and 30 minutes in mock (□) and KLF2 (▦) lentivirus-transduced HUVECs. (C) The amount of RalA 7 days after transduction of HUVECs with mock and KLF2 lentivirus quantified from Western blots (n = 4). (D) Dual immunofluorescence for VWF (red) and RalA (green) in HUVECs 7 days after transduction with KLF2 lentivirus. Colocalization of RalA with VWF is shown in yellow. Image was acquired using a 100 ×/1.5 NA oil objective. (E) Time course of the regulated VWF secretion after thrombin (circles) or forskolin (squares) stimulation of Lenti-mock (open symbols) and Lenti-KLF2 (filled symbols) HUVEC cultures. (F) The number of WBPs in VWF-expressing cells of mock-(circles) and KLF2-transduced (diamonds) HUVEC cultures was determined with (open symbols) or without (filled symbols) prior stimulation with thrombin or forskolin for 30 minutes. (G-H) Stimulated VWF secretion was measured by enzyme-linked immunosorbent assay (ELISA) following a 30-minute exposure of Lenti-mock (○) and Lenti-KLF2 (•) transduced HUVECs to increasing concentrations of epinephrine (G) and thrombin (H). *P < .01 and **P < .001.