Figure 1: NME overexpression increased tumor cell endocytosis.

Previously reported transfectants of the MDA-MB-231T breast cancer cell line expressed a vector construct or NME genes (human NME1, NME2 or mouse ortholog Nme1), while transfectants of the MDA-MB-435 cell line expressed a vector (C-100) or NME1 (H1–177). A-D MDA-MB-231T transfectants were incubated with Transferrin-Alexa-594 (A) or pHrodo™ Red-EGF (C) (both red) for 15 min, whereupon cells were fixed in PFA and nuclei stained with DAPI (blue). Representative merged images are shown at 63x magnification. Scale bar-10 μm. Lysates of the transfectants were probed for the cognate receptors on western blots, Transferrin receptor (B) and EGFR (D). β-actin was used as normalizing control. E, F MDA-MB-435 transfectants similarly incubated with Transferrin-Alexa-594 (E) or pHrodo™ Red-EGF (F). G Western blot of MDA-MB-435 cells for the cognate receptors. H Vector and NME1 overexpressing MDA-MB-231T cells were incubated with pHrodo™ Red-EGF dye (red) for 15 min followed by immunostaining with Rab5 antibody (far-red). Approximately 32% of pHrodo™ Red-EGF co-localized with Rab5 in NME1 overexpressing cells (white). Scale bar-10 μm. I Immunofluorescence staining of vector and NME overexpressing MDA-MB-231T cells for AP2 alpha to detect clathrin coated pits (red). Cells are outlined in dashed lines with nuclei stained with DAPI (magnification-100x). J Rab5-GTP levels determined by active Rab5 Pull-down assay from whole cell lysates of vector and NME overexpressing MDA-MB-231T cells. Upper panel: Rab5-GTP, Lower panel, Total Rab 5. K Time lapse real time imaging was performed on vector and NME overexpressing (NME1 and NME2) MDA-MB-231T cells incubated with pHrodo™ Red-EGF dye (Supplementary Video S1). Frames at 1, 5, 10 and 15 min are shown. Data are representative of three independent experiments.