Fig 1: Enhanced EGFR endocytosis due to PLD2 overexpressionA. Endogenous EGFR intracellular localization following EGF stimulation. MTLn3 cells were serum-starved and then treated with 10 nM EGF for the times indicated. EGFR was stained with a-EGFR-FITC IgG antibodies (left column of panels), while proteins specific to the different organelles (cell membrane, Cadherin; early endosomes, EEA1; late endosomes, Rab7; recycling vesicles, Rab11 or Golgi, RCAS1) were stained with anti-TRITC IgG antibodies (2nd column from the left). Nuclei were stained with DAPI (blue) (2nd column from the right). B. After PLD2 overexpression, EGFR was stained with a-EGFR-FITC IgG antibodies (green staining), while antibodies specific to PLD2 were stained with anti-TRITC IgG antibodies (red staining). Nuclei were stained with DAPI (blue). Merged images of overlaid FITC, TRITC and DAPI images are shown in right panels. Shown are images representative of n = 3 independent experiments, with visualization of 5 fields in each condition, with similar results. C.-D. Cells were serum-starved and then treated with 10 nM EGF for the indicated times. Proteins specific to different cellular organelles were as in the previous panel. TRITC images were inverted from color to black/white and then blue DAPI images were merged together using Adobe PhotoShop (all panels as shown). For (A-D), images are representative of n = 3 of separate experiments after visualizing 5 separate fileds. Scale bar = 10 µm. E.-F. Quantification of the total number of punctae per cell (>0.1 µm) for either early endosomal EEA1 (E) or Golgi RCAS1 (F) staining following EGF treatment in the absence or presence of PLD2 overexpression, respectively. The (*) symbols above bars denote statistically significant (P < 0.05) ANOVA increases between samples and controls. The (#) symbols above bars denote statistically significant (P < 0.05) ANOVA decreases between samples and controls.