Co-localization between PrP^Sc and different sub-cellular organelles in TNT-like structures of ScCAD cells. (A) Representative immunofluorescence of PrP from CAD and ScCAD after guanidium thiocyanate denaturation. Images were acquired at exactly the same acquisition parameters. PrP^Sc puncta are clearly revealed by enhanced epitope detection after denaturation while PrP^C signal is diminished. Scale bar: 10 μm. (B) Co-immunofluorescence for different organelle markers and PrP^Sc was performed after guanidium thiocyanate treatment to determine colocalization. Scale bar: 5 μm. (C) Quantification of the colocalization between PrP^Sc and the different organelle markers in TNT structures. The percentage of PrP^Sc particles co-localizing respectively with EEA1 (for early endosomes), Lamp1 (for lysosomes) and Vamp3 (ERC) was evaluated. (Mean + s.e.m).

Effect of PrP^Sc and PrP^C on TNT formation and intercellular vesicle transfer. (A) Representative image of TNT formation in uninfected CAD cells (top panel) and chronically prion-infected ScCAD (bottom panel). In green, the plasma membrane labeled with WGA488 and red, the nucleus. (B) Quantification of TNT formation in these 2 cell lines show that prion infection increases TNT forming cells by ˜20% (p = 0.0393, one tailed t-test respectively). (C) Prion infection increases DiD-labeled vesicle transfer to mCherry-H2B-labeled acceptor cells (p= 0.0429). Medium CAD and medium ScCAD respectively indicate mCherry-H2B-labeled CAD cells that had been treated with medium from either DiD-labeled CAD or ScCAD as described in methods. Transfer does not seem to be secretion-mediated. (D) Representative image of TNT formation from cells overexpressing either GFP-vector (top panel) or GFP- PrP^C (bottom panel). In green: GFP or GFP- PrP^C respectively; in red: the plasma membrane labeled with WGA-546 and H2B-mCherry-labeled nuclei of acceptor cells. (E) Quantification of percentage of TNT forming cells in CAD overexpressing GFP or GFP- PrP^C. GFP-PrP overexpression increases TNT forming cells (p = 0.05). (F) Quantification of the percentage of acceptor cells which contain transferred DiD-labeled vesicles after overnight co-culture shows no significant difference between GFP- or GFP- PrP^C expressing cells. Scale bars, 10μm. For all graphs, the total number of cells counted (n) over 3 independent experiments is given on each respective column.