American Society of Hematology

Figure 3

$Figure 3. Nystatin switches endostatin internalization from caveolae/lipid rafts to clathrin-coated pits. (A) HUVECs were treated with 0 or 25 μg/mL nystatin, incubated with 5 μg/mL endostatin at 37°C for 30 minutes, and lysed with 1% Triton X-100 on ice for 30 minutes. Triton X-100 flotation gradients were prepared with a 5%/30%/40% OptiPrep gradient. Clathrin heavy chain (Clathrin HC) and transferrin receptor (Transferrin R) were used as markers for nonraft fractions. Cav1 and glypican-1 were used as markers for raft fractions. (B) HUVECs were treated with 0 or 25 μg/mL nystatin, then incubated with 2 μg/mL FITC-CTB and 5 μg/mL Rh-endostatin at 37°C for 30 minutes. Cells were further fixed and examined by a confocal microscope. (C-D) HUVECs were transfected with GFP-Cav1 or GFP-clathrin light chain (CLC) for 48 hours. Cells were then treated with 0 or 25 μg/mL nystatin and incubated with 5 μg/mL Rh-endostatin at 37°C for 30 minutes. (E) The levels of colocalization (B-D) were quantified by calculating the percentage of red pixels (Rh-endostatin) that colocalized with green pixels (FITC-CTB, GFP-Cav1, or GFP-CLC) from ≥ 8 random fields per well of 3 experiments. (F) Triple fluorescent staining with GFP-CLC (green), Rh-endostatin (red), and anti-Cav1 primary Ab followed by a cyanine 5–conjugated secondary Ab (blue) in the absence or presence of nystatin. Images were obtained using a Nikon A1 microscope (60×/1.40 NA oil objective). Scale bar, 10 μm. **P < .01. Error bars represent SEMs.$

Nystatin switches endostatin internalization from caveolae/lipid rafts to clathrin-coated pits. (A) HUVECs were treated with 0 or 25 μg/mL nystatin, incubated with 5 μg/mL endostatin at 37°C for 30 minutes, and lysed with 1% Triton X-100 on ice for 30 minutes. Triton X-100 flotation gradients were prepared with a 5%/30%/40% OptiPrep gradient. Clathrin heavy chain (Clathrin HC) and transferrin receptor (Transferrin R) were used as markers for nonraft fractions. Cav1 and glypican-1 were used as markers for raft fractions. (B) HUVECs were treated with 0 or 25 μg/mL nystatin, then incubated with 2 μg/mL FITC-CTB and 5 μg/mL Rh-endostatin at 37°C for 30 minutes. Cells were further fixed and examined by a confocal microscope. (C-D) HUVECs were transfected with GFP-Cav1 or GFP-clathrin light chain (CLC) for 48 hours. Cells were then treated with 0 or 25 μg/mL nystatin and incubated with 5 μg/mL Rh-endostatin at 37°C for 30 minutes. (E) The levels of colocalization (B-D) were quantified by calculating the percentage of red pixels (Rh-endostatin) that colocalized with green pixels (FITC-CTB, GFP-Cav1, or GFP-CLC) from ≥ 8 random fields per well of 3 experiments. (F) Triple fluorescent staining with GFP-CLC (green), Rh-endostatin (red), and anti-Cav1 primary Ab followed by a cyanine 5–conjugated secondary Ab (blue) in the absence or presence of nystatin. Images were obtained using a Nikon A1 microscope (60×/1.40 NA oil objective). Scale bar, 10 μm. **P < .01. Error bars represent SEMs.

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