DF is internalized by ECs through macropinocytic mechanisms. (A) Bar diagram shows the decrease in the uptake of DF by SK cells in the presence of endocytosis and vesicle-trafficking inhibitors. Data obtained from flow cytometry experiments are expressed as mean ± standard error of the mean, n = 6, being *P < .05 vs 100% of positive cells for DF in the absence of the inhibitors. (B) Confocal microscopy images correspond to the negative results of colocalization assays between DF (green, labeled with Alexa 488) and clathrin, caveoline, and lysosomes (first, second, and third lines, respectively). SK cells were incubated with DF for 15 minutes to evaluate colocalization with clathrin and caveoline, and for 6 hours to evaluate DF–lysosomal interaction. (C) Images to the left and right correspond to SK cells incubated with DF (green) in the absence or presence of Wortmannin (W), respectively (red staining with wheat germ agglutinin for membranes). Graphs above represent mean fluorescence intensity and follow the same distribution. DF staining inside the cells can be visualized in the absence of W (left image, left graphic). DF staining is attached to the membrane in the presence of W (right image, right graphic). Confocal images were taken using a Leica TCS SP5 microscope and a 63× oil immersion objective. Image analysis was performed using Fiji software (National Institutes of Health).