Colocalization of rNadA with endosome markers.

A: Chang cells were incubated with 200 µg/ml rNadA for 1 h at 37°C, then fixed, permeabilized and double stained for rNadA (green) and EEA1 (A), Rab5 (B), M6PR (C), Clathrin (D) and TfR (E) (red). Merged images of the red and green signals are also shown. Images are representative of two independent experiments. Scale bar is 10 µm. F: Quantification of rNadA colocalization with endosome markers. The black columns indicate the percentage of rNadA immunofluorescent pixels colocalizing with the endosomal marker (EEA1, RAb5, M6PR, Clathrin, TfR as indicated) immunofluorescent pixels. Viceversa the grey columns indicate the percentage of the endosomal marker immunofluorescent pixels colocalizing with rNadA immunofluorescent pixels. Data are mean ± s.e.m representative of two independent experiments, each assessing 20–25 cells. G: Chang cells were transfected with an EGFR-AP180C expressing plasmid or with empty vector and incubated 24 hours at 37°C to recover. Cells were then incubated with rNadA, fixed and stained as indicated in point A. EGFR-AP190C is shown in green, rNadA in red and TfR in blu. Quantification was performed as indicated in point F.