Effects of caveolae pathway or clathrin pathway or both pathway inhibition on endostatin internalization in endothelial cells. (A-B) HUVECs were treated with 0, 3, or 6 μg/mL chlorpromazine together with or without nystatin, then applied to endostatin internalization assay. (C) HUVECs were transfected with siRNA against Cav1 (si-Cav1), CHC (si-CHC), or both (si-Cav1-CHC) for 48 hours. Then endostatin internalization was assessed in the absence or presence of nystatin treatment. (D-F) HUVECs were transfected with GFP-tagged Eps15-DIII or wild-type control (Eps15-DIIIΔ2), then treated with 0 or 25 μg/mL nystatin before endostatin incubation. Endostatin internalization was analyzed by immunofluorescence using a Nikon A1 microscope (60×/1.40 NA oil objective) (D-E) and immunoblotting (F). Biotin-transferrin was a marker for clathrin-mediated endocytosis and was detected by the avidin-biotin immunoperoxidase method. Scale bar, 20 μm.