Trypsin treatment

When the cell confluence reached 60%, colchicine (Sigma-Aldrich, Burlington, MA, USA) was added into the culture medium. Five hours later, cells were collected and centrifuged at 1500 rpm for 10 min. After being resuspended in 0.075 mol/L potassium chloride (KCl) solution, the cells were incubated at 37 °C for 20 min, immobilized by Carnoy fixative and dried on clean glass slides. G-bands of chromosomes were visualized using trypsin treatment and Giemsa staining (Sigma-Aldrich, Burlington, MA, USA).

REFs were prepared from Wt rats at E14.5. Embryos were dissociated by trypsin treatment (Sigma-Aldrich), and the single cells were cultured in DMEM including 10% FBS and antibiotic-antimycotic solution at 37 °C in 5% CO₂. Serum-derived EVs from Wt or Tg rats were isolated using ultracentrifugation. The EVs were visualized using a PKH67 fluorescence labelling kit (Sigma-Aldrich). Serum EVs were incubated with REFs on 24-well glass bottom plates for 10–11 hours at 37 °C in 5% CO₂. After being washed, the cultured cells were fixed with 4% PFA. The EVs from Tg rats were detected using anti-human CD63 antibody after having been transferred into REFs. To analyse localization of EVs in REFs, anti-LAMP1 antibody (1:250, Sigma-Aldrich) was used. After the cells were stained with Alexa Fluor 546 Goat anti-Mouse IgG₁ (1:2000; Life Technologies), Alexa Fluor 546 Goat anti-Rabbit IgG (1:2000; Life Technologies) and Alexa Fluor 488 Goat anti-Rabbit IgG (1:200; Life Technologies) antybodies, internalization of EVs were captured with a FV1000 confocal microscope (OLYMPUS, Tokyo, Japan) and a BIOREVO BZ-9000 fluorescence microscopy (Keyence, Osaka, Japan). Hoechst 33342 was used to stain cell nuclei.
All experimental protocols were approved by the National Institute of Neuroscience, National Center of Neurology and Psychiatry, and National Cancer Centre Research Institute, Japan.