Cholesterol sequestration increases endostatin internalization in endothelial cells. (A-B) HUVECs were treated with increasing concentrations (0-100 μg/mL) of nystatin (Nys) for 30 minutes or 25 μg/mL nystatin for increasing time periods (0-30 minutes). Then the cells were incubated with 5 μg/mL endostatin at 37°C for 30 minutes, washed with acidic buffer, and examined for internalized endostatin by immunoblotting. (C) Immunoblot analysis of internalized endostatin levels in whole-cell lysate (WCL), cytosol (Cyt), and nucleus (Nuc). Lamin B and β-actin were used as markers for nuclear and cytoplasm, respectively. (D) HUVECs were resupplied with cholesterol for 30 minutes after nystatin treatment (25 μg/mL, 30 minutes), then applied to endostatin internalization assay. (E) Flow cytometric histogram of Rh-endostatin internalization at 37°C (20 μg/mL, 30 minutes) with 0, 10, or 25 μg/mL nystatin treatment. (F) Mean fluorescence intensity of Rh-endostatin internalization histogram analysis from 3 independent experiments. (G) Immunofluorescence detection of Rh-endostatin in nystatin-treated HUVECs using a Nikon A1 microscope (60×/1.40 NA oil objective). (H) Internalized Rh-endostatin was quantified as the mean fluorescence intensity from ≥ 8 random fields per well of 3 experiments. Scale bar, 10 μm. *P < .05, **P < .01, and ***P < .001. Error bars represent SEMs. P values were calculated with the Student t test.