FIGURE 5.

PMPs are actively ingested and transported to the nuclear vicinity. HAECs were grown on coverslips and incubated with fluorescent PMPs in the presence and absence of Gas6 for 4 h. Cells were fixed, and the cell membrane was stained with an antibody against CD31, after which uptake was evaluated using confocal microscopy. CD31 is shown in red; PMPs are shown in green, and the nuclei are shown in blue (A). Uptake of Gas6-coated biotinylated PMPs was investigated using transmission electron microscopy. Cells were stained with an avidin-gold conjugate to visualize PMPs. Arrows point to PMPs moving freely in the cell and arrowheads to PMPs enclosed in endosome-like compartments (B). Scale bars, 20 μm (A) and 500 nm (B). HAECs were treated with cytochalasin D or nocodazole prior to and during uptake of fluorescent PMPs in the presence and absence of Gas6. Uptake was measured by flow cytometry (C). Successful disruption of the microtubule and the actin networks was verified by staining the cells with anti-β-tubulin and fluorescent phalloidin (D). Data in A show representative images from at least three individual experiments, and C shows the mean and S.D. of three separate experiments. B shows two out of several representative images from one uptake experiment. ****, p < 0.0001.