PTX2 reduces FX internalization in SR-AI–expressing HEK293 cells. SR-AI–expressing HEK293 cells were incubated with 150 nM of FX (A), 500 nM of PTX2 (B), or both (C) for 1 hour at 37°C and then washed and fixed. Representative confocal microscopy images of FX or PTX2 immunostaining along with SR-AI (A-B) or coimmunostaining of FX and PTX2 (C) are depicted. FX (red) and EEA-1 (green) were immunostained in SR-AI–expressing HEK293 cells incubated with either FX (D) or FX + PTX2 (E), and images were acquired in confocal microscopy. FX colocalization with EEA-1 was quantified by calculating the tMC for FX using JACoP plugin in Fiji software (F). Results are expressed in a whisker plot where boxes represent the median and 25th to 75th percentile, and bars represent the minimum and maximum of 10 to 11 cells in 3 different experiments. *P < .05; ***P < .001 in a Mann-Whitney nonparametric unpaired statistical test. N.S., not significant. In representative confocal images, dotted lines define cell boundaries based on phalloïdin staining, arrows indicate internalized FX or PTX2 colocalized with SR-AI (A-B) or area of colocalization between FX and SR-AI (D), and arrowheads indicate colocalization between FX and PTX2 (C). Objective 63×, z-depth 1 µm; bars represent 10 µm.