Figure 3: ARF6 regulates CD63 late endosomal trafficking.

(a) MCF-7 cells were treated with ARF6#1 RNAi (siARF6) or non-targeting RNAi (siCNT). The internalization of the CD63 antibody was monitored by confocal microscopy analysis after 0, 15 and 30 min. The fraction of total CD63 internalized was measured using 10 to 20 cells, taken from two independent experiments (see Supplementary Fig. 3a,b for illustrations). The graph illustrates the mean percentage of intracellular CD63 signal, the bars correspond to s.d. (b) Representative confocal micrographs showing the steady-state subcellular distribution of endogenous CD63 together with that of endogenous EEA1, overexpressed eYFP-RAB11 or endogenous LAMP2, in CNT cells and in ARF6-depleted cells (ARF6#2 RNAi). See insets for high magnification ( × 3). Note the high level of co-localization of CD63 with LAMP2, not with EEA1 or eYFP-RAB11 in ARF6-depleted cells. In the merge, CD63 is in red, nuclei were stained with Hoechst (blue), and endosomal markers are in green. Scale bar, 10 μm. (c) CD63 colocalization with EEA1, RAB11 and LAMP2 was assessed by calculating the Pearson correlation coefficient on 10 cells per condition using the JACoP plugin on ImageJ. Histograms represent the mean Pearson coefficient±s.d. in ARF6-depleted cells, relative to signals in siCNT cells, calculated for three independent experiments. **P<0.01 (Student’s t-test). (d) Representative electron micrographs, illustrating the morphology of the late endosomal compartment in MCF-7 cells treated with control, syntenin or 2 different ARF6 RNAi, as indicated. Structures were revealed by peroxidase-conjugated anti-CD63 internalized for 30 min, and staining with DAB. Scale bar, 200 nm. (e) Endosome filling. For each peroxidase/DAB-marked endosome, the stained sectional area of the endosome is plotted against the total sectional area of that endosome. siARF6#1 and siARF6#2 data were pooled, n is the total number of endosomes examined in four independent experiments.