Figure 2

(a) Representative confocal images of 4T1 cells co-transfected with PLD1-GFP and tdTomato-RalA (upper panels) or tdTomato-RalB (lower panels) and incubated with Lysotracker. Scale bar: 10 μm; zoom: 2 μm. (b) Electron microscopy analysis of 4T1 cells treated with PLD1 or PLD2 inhibitor. Scale bar: 1 μm. Violin plots show quantification of the number of multi-vesicular body (MVB) per cytoplasmic surface. Each dot represents one field of view; horizontal bar represents the average (180–194 fields of view; Kruskal-Wallis test followed by Dunn's Multiple Comparison Test). (c) Nanoparticle tracking analysis of extracellular vesicles (EVs) isolated by ultracentrifugation (100,000 g pellet) from the supernatant of 4T1 cells treated with PLD1 (CAY10593) or PLD2 (CAY10594) inhibitor. Each dot represents one experiment (three independent experiments; One-Way Anova permutation test followed by fdr multi-comparison permutation test). (d) Representative confocal images of shControl, shRalA and shRalB 4T1 cells transfected with PLD1-GFP. Scale bar: 10 μm; zoom: 2 μm. Graph shows the percentage of cells with high (>5) number of PLD1-GFP cytoplasmic puncta. (Each dot represents one experiment. Five independent experiments; Number of cells analyzed: shCtl (136), shRalA (170), shRalB (244); Kruskal-Wallis test followed by Dunn's Multiple Comparison Test). (e) Quantification of the Phosphatidic Acid (PA) / PhosphatidylCholine (PC) ratio in EVs isolated from shControl, shRalA, and shRalB cells (each dot represents one experiment; three independent experiments; One-Way Anova permutation test followed by fdr multi-comparison permutation test; fdr <0.1). (f) Model showing how RalA and RalB could control PLD1 localization on MVBs, thereby inducing the PA accumulation on MVBs, promoting MVB homeostasis and controlling exosome secretion.