GM1 is responsible for the binding of sEVs to laminin. (A) (top) Schematic diagram of gangliosides and (bottom) chemical structure of GM1. Monosaccharide symbols follow the Symbol Nomenclature for Glycans (SNFG). (B) Dot blotting of GM1 and GM3 in PC3 cells and PC3-derived sEVs. (C) Western blot analysis of integrin subunits in B78 cell lines with abundant expression of one type of ganglioside—GM1, GM2, GM3, GD2, or GD2/GD3—and sEVs derived from these cells. (D and E) The numbers of sEVs attached to glass coated with laminin (D) and the numbers normalized to the ratio of integrin α6/CD81 in the sEVs (E). (F and G) The numbers of sEVs attached to glass coated with fibronectin (F) and the numbers normalized to the ratio of integrin α5/CD81 in the sEVs (G). (H) Single-particle fluorescence images of DMPC liposomes containing GM1, GM2, GM3, GD1a, GD2, or GD3 on glass coated with fibronectin or laminin. (I and J) The numbers of liposomes attached to glass coated with laminin (I) or fibronectin (J). (K) Single-particle fluorescence images of sEVs-PC3-CD63Halo7-TMR and sEVs-PC3-ITGB1KO-CD63Halo7-TMR on laminin before or after treatment of the GM1’s glycan. (L) The numbers of sEVs bound to laminin before or after treatment with a high concentration of the GM1 glycan moiety (0.5 mM final). Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided). In I and L, due to the necessity of multiple statistical tests, the significance level was corrected by the Bonferroni method and determined to be 0.025 (=0.05/2). Source data are available for this figure: SourceData FS4.