CD151 and cholesterol regulate the binding affinity of sEVs for laminin. (A) Western blot analysis of CD151 and integrin subunits in PC3 cells and sEVs after CD151 KD. The amount of cell proteins loaded in one lane was 2.5 times greater than that of the sEVs in the other lane. (B) Images of WT PC3 cells and CD151 KD cells on glass coated with ECM components (fibronectin [FN], laminin [LN], or collagen type Ⅰ [COL1]) after 2 h of incubation. The areas of the cells were quantified (n = 20 cells). (C) Single-particle fluorescence images of sEV–CD63Halo7-SF650T particles bound to laminin (LN) on glass before and after CD151 was knocked down and cholesterol was depleted by MβCD (left). The number of attached sEVs increased (right) (n = 21 images). (D) The binding affinity ratio of cholesterol-depleted sEVs to intact sEVs was compared with that of CD151 KD sEVs. (E) Single-particle fluorescence images and the number of sEVs attached to collagen type I on glass before and after CD151 KD (n = 19 images). (F) Western blot analysis of integrin–CD151 complex in PC3-derived sEVs before and after treatment with MβCD. The complex was immunoprecipitated with anti-integrin β1 and blotted with anti-integrin α6, α3, or CD151 antibodies. Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided). In C, 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 F8.