PLD stimulates the formation of lipid rafts in a dose-dependent manner. HeLa cells were treated (A) without or (B) with 50 ng PLD for 10 min at 37°C followed by staining with the Vybrant Lipid Raft Labeling Kit. The arrows indicate cells with bright, punctate staining, while the arrow heads indicate diffusely staining cells. Bar 50 μm. (C) HIS-PLD was added to HeLa cells for 10 min at 37°C prior to measurement of lipid raft formation. At least 100 cells were counted and the percentage of cells displaying punctate staining were enumerated. (D) Anti-PLD antibodies or the cholesterol sequestering agent MβCD inhibit PLD-mediated lipid raft formation. HeLa cells were untreated, or treated with 1/1000 dilutions of pre-immune or anti-PLD serum or 5 mM MβCD prior to addition of 50 ng HIS-PLD and measurement of lipid raft formation. Untreated HeLa cells or those treated with pre-immune, anti-PLD serum or MβCD, but not HIS-PLD served as the negative controls. Error bars indicate one standard deviation from the mean calculated from the averages of at least three independent experiments conducted in triplicate. Statistical significance was calculated using single factor ANOVA and p < 0.05 was considered significant.