Fig 2: Rac1 and AKT are responsible for phosphorylation of β-catenin in ECsA. Phosphorylation of β-catenin in presence of Akt inhibitor. E4+ECs, serum-starved for 24 h, were pre-treated or not with LY 294002 (10μM). Phosphorylation of β-catenin Ser675 and Ser552 and of Akt at Ser473 were analyzed by western blot. While LY 294002 was able to inhibit the phosphorylation of Akt, it was only able to reduce the phosphorylation of β-catenin Ser552 but not Ser675. B. Rac1 activation. E4+ECs, serum-starved for 24 h, were treated with OCC-MPs for 15 or 30 minutes. Western blot analysis revealed an increase of Rac1 after incubation with OCC-MPs. C. Phosphorylation of β-catenin in the presence of Rac1 inhibitor. E4+ECs, serum-starved for 24 h, were pre-treated or not with NSC23766 (10μM). Phosphorylation of β-catenin Ser675 and Ser552 were analyzed by western blot. NSC23766 was able to reduce the phosphorylation of β-catenin at Ser552 and at Ser675. D. Phosphorylation of β-catenin in the presence of PAK1 blocking antibody. E4+ECs, serum-starved for 24 h, were pre-treated or not with a blocking antibody for PAK1. Phosphorylation of β-catenin Ser675 and Ser552 were analyzed by western blot. The inhibition of PAK1 reduced the phosphorylation of β-catenin Ser552 and Ser675. E. OCC-MPs uptake by E4+ECs in presence of inhibitor. MPs from APOCC or Skov3 were extracted from 80% confluent cells and labelled with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). MPs uptake by eGFP-E4+ECs was quantified by flow cytometry in absence (purple and green plot) or presence of LY294002 or NSC23766 (orange plot). The inhibitors of AKT and Rac1 were not able to modify OCC-MPs uptake by E4+ECs. F. β-catenin localization. E4+ECs pre-treated or not with LY294002 and NSC23766, were treated for 1h with the MPs extracted from Skov3, APOCC. Staining for β-catenin was performed and localization of β-catenin was studied by confocal microscopy. When E4+ECs were pre-treated with LY294002 and NSC23766, β-catenin was not translocated to the nucleus but accumulated in the cytoplasm. Scale bar: 10 μm.

Fig 3: Rho-GTPase signalling supports MB cell migration.A Representative photographs of immunofluorescence analysis performed in DAOY, UW228-2, D425, D458 and ICb-1299 cells using antibodies against anti-Farnesyl and RhoA/B/C proteins. Nucleus was stained with DAPI (blue). Scale bar = 20 µm. Quantification of mean fluorescence intensities from 3 independent experiments of anti-Farnesyl (B) and Rho A/B/C (C) in DAOY, UW228-2, D425, D458 and ICb-1299 cells performed with GraphPad Prism. One-way ANOVA performed with Tukey’s post-Hoc comparisons, where ** = P < 0.01 and **** = P < 0.0001. D, E Western blotting showing Rho A levels in SHH-MB (DAOY, UW228-2) vs G3-MBs (D425, D458) cells. Vinculin was used as a loading control. Fold change in protein levels between SHH cells and G3/G4 MBs cells is displayed. F Quantification of mean fluorescence intensities from three independent experiments of anti-Farnesyl in DAOY, UW228-2, D425, D458 and ICb-1299 cells after a single treatment with 5 μM simvastatin. Simvastatin treatment was performed for 2, 6, 16 and 24 h.