Fig. 2

PtdSer internalization is ATP independent. a Representative images of HeLa cells expressing GFP-LactC2 treated with 10 mM mβCD for 30 min with or without ATP depletion, which was carried out pretreating cells with 0.2 µM antimycin A and 10 mM 2-deoxy-D-glucose for 60 min in Ca²⁺-free and glucose-free medium. After mβCD treatment, cells were labeled with 40 ng/µL FM4-64. b Cholesterol extraction using mβCD was performed at 4 °C or 37 °C, as indicated, followed by fixation with 4% paraformaldehyde and labeling with 0.05% filipin. Inset: control cells without cholesterol removal. c mβCD was removed after 30 min and GFP-LactC2-expressing cells were labeled with FM4-64. Inset: magnifications of the area indicated by a dashed square. Scale bar = 10 µm in all images