Fig.3

Analysis of AQP4 distribution in EVs released from GBM U87 cells. a Immunoblot detection of AQP4 expression levels in cell lysates (L) and pelleting fractions (300 g, 2 K, 10 K and 100 K) derived from conditioned media of U87 transfected with the AQP4-tetramers (M1) or the AQP4-OAPs (M23) isoform as indicated in each lane. Flotillin-2 is used as a marker of large EVs (2 K pellet) and CD81 as an exosomal marker (100 K pellet). b Densitometric analysis of the immunoblot in a showing AQP4-M1 and AQP4-M23 enrichment in EVs subtypes. Values are expressed as the ratio of AQP4 expression in the fractions /AQP4 total expression (%) ± SEM ***P < 0.0005, n = 3, two-way ANOVA/Tukey’s test. c Epifluorescence images of U87 WT used as recipient cells treated with 2 K and 10 K EVs-derived from U87 transfected with empty vector (Mock), AQP4-tetramers or AQP4-OAPs after 24 h of incubation. Recipient cell membrane is stained with WGA (red), while EVs are stained in green for AQP4. DAPI for nuclear staining is in blue. The arrowheads showing perinuclear localization of EVs in recipient cells. Scale bar 50 μm. d Single optical intracellular plane of 3D confocal reconstruction and relative YZ and XZ slices showing internalization of large (2 K) and small (10 K) EVs in recipient cells. Recipient cell membranes are stained with WGA (red), AQP4 staining is shown in green. The arrows indicate internalization or interaction of EVs with plasma membrane of recipient cells. Scale bar 20 μm