Fig 3. Silencing of RAB8B, RAB13 or SYTL5 increases aSyn cell-to-cell trafficking.

A. Experimental design of aSyn cell-to-cell trafficking assay. Stable cells silenced for RAB8B, RAB11A, RAB13, SYTL5 or with a scrambled shRNA were transfected separately with plasmids encoding either VENUS1-aSyn or aSyn-VENUS2. 24 h later, cells were mixed and co-cultured for 72 h. Upon release and uptake of the aSyn-VENUS fusions, reconstitution of the fluorescence signal can be detected inside cells, and the signal quantified through flow cytometry or microscopy. B. VENUS positive cells were monitored by flow cytometry. A representative result is shown as side scatter (SSC) versus VENUS fluorescence, with the corresponding histogram. The percentage of VENUS positive cells is indicated by the mean±95% CI of at least three independent experiments. C. In vivo imaging of aSyn-VENUS1 and VENUS2-aSyn mixed cells subjected to silencing of the selected hits. Scale bar: 20 μm. D. Immunoblotting analysis of, total aSyn and beta-actin. Bars represent mean±95% CI (*: 0.05<p>0.01; **: 0.01<p>0.001; ***: p<0.001) and are normalized to the control of at least three independent experiments. Single comparisons between the control and experimental groups were made through Wilcoxon test. kd, knockdown.