Figure 4.
Integrins β1 and α6 in sEVs and the ECM are responsible for the binding of sEVs to cell PMs. (A) Immunofluorescence images of fibronectin, collagen type I, laminin, laminin α5, and laminin α1 in human normal embryonic lung fibroblast (MRC-5) cells and human bone marrow stromal (HS-5) cells. (B) The fluorescence intensities of laminin on cells in 1,000 μm2 (n = 4 images). (C) TIRF images of MRC-5 cells expressing mCherry and sEV–CD63Halo7-SF650T particles (arrowhead) attached to cell membranes after 10, 20, and 30 min of incubation. sEVs were isolated from the cell culture supernatant of intact PC3 cells (top panels) and from the cell culture supernatant of integrin β1 KO PC3 cells (bottom panels) bound to the basal surface of MRC-5 cells. (D) TIRF images of HS-5 cells and sEV–CD63Halo7-SF650T particles (arrowhead) attached to the basal surface of cell membranes after 30 min of incubation. sEVs were isolated from intact PC3 cells (top panels) and integrin β1 KO PC3 cells (bottom panels). (E) TIRF images of MRC-5 cells and the attached sEV–CD63Halo7-SF650T particles derived from intact PC3 cells (top panels) and from integrin α6 KO PC3 cells (bottom panels) after 30 min of incubation. (F–H) Time course of the numbers of intact sEVs and integrin β1 KO sEVs attached to the basal surface of the MRC-5 cell membrane (n = 8 images) (F) and to the basal surface of the HS-5 cell membrane (n = 15 images) (G) per 1,000 μm2. (H) Time course of the numbers of intact sEVs and integrin α6 KO sEVs attached to the basal surface of the MRC-5 cell membrane per 1,000 mm2 (n = 12 images). Data are presented as the mean ± SE. (I) Fluorescence images of MRC-5 cells expressing GFP and sEV–CD63Halo7-TMR by confocal microscopy. sEVs were isolated from the cell culture supernatant of WT PC3 cells (top panels) and from that of integrin β1 KO PC3 cells (bottom panels). The cells were fixed after treatment of the sEVs for 1 h. (J) The numbers of sEVs attached to MRC-5 and HS-5 cell PMs by confocal fluorescence microscopy (n = 10 images). In Fig. 4, since not all cells necessarily express mCherry or GFP, many fluorescent spots of sEVs were observed either in regions containing non-expression cells or potentially on the glass surface. Nevertheless, we can quantify the number of sEVs bound to both the apical and basal surfaces of the cell PM by counting the number of fluorescent EV spots on cells expressing mCherry or GFP. Data are presented as the mean ± SE. n.s., nonsignificant difference; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided).

Integrins β1 and α6 in sEVs and the ECM are responsible for the binding of sEVs to cell PMs. (A) Immunofluorescence images of fibronectin, collagen type I, laminin, laminin α5, and laminin α1 in human normal embryonic lung fibroblast (MRC-5) cells and human bone marrow stromal (HS-5) cells. (B) The fluorescence intensities of laminin on cells in 1,000 μm2 (n = 4 images). (C) TIRF images of MRC-5 cells expressing mCherry and sEV–CD63Halo7-SF650T particles (arrowhead) attached to cell membranes after 10, 20, and 30 min of incubation. sEVs were isolated from the cell culture supernatant of intact PC3 cells (top panels) and from the cell culture supernatant of integrin β1 KO PC3 cells (bottom panels) bound to the basal surface of MRC-5 cells. (D) TIRF images of HS-5 cells and sEV–CD63Halo7-SF650T particles (arrowhead) attached to the basal surface of cell membranes after 30 min of incubation. sEVs were isolated from intact PC3 cells (top panels) and integrin β1 KO PC3 cells (bottom panels). (E) TIRF images of MRC-5 cells and the attached sEV–CD63Halo7-SF650T particles derived from intact PC3 cells (top panels) and from integrin α6 KO PC3 cells (bottom panels) after 30 min of incubation. (F–H) Time course of the numbers of intact sEVs and integrin β1 KO sEVs attached to the basal surface of the MRC-5 cell membrane (n = 8 images) (F) and to the basal surface of the HS-5 cell membrane (n = 15 images) (G) per 1,000 μm2. (H) Time course of the numbers of intact sEVs and integrin α6 KO sEVs attached to the basal surface of the MRC-5 cell membrane per 1,000 mm2 (n = 12 images). Data are presented as the mean ± SE. (I) Fluorescence images of MRC-5 cells expressing GFP and sEV–CD63Halo7-TMR by confocal microscopy. sEVs were isolated from the cell culture supernatant of WT PC3 cells (top panels) and from that of integrin β1 KO PC3 cells (bottom panels). The cells were fixed after treatment of the sEVs for 1 h. (J) The numbers of sEVs attached to MRC-5 and HS-5 cell PMs by confocal fluorescence microscopy (n = 10 images). In Fig. 4, since not all cells necessarily express mCherry or GFP, many fluorescent spots of sEVs were observed either in regions containing non-expression cells or potentially on the glass surface. Nevertheless, we can quantify the number of sEVs bound to both the apical and basal surfaces of the cell PM by counting the number of fluorescent EV spots on cells expressing mCherry or GFP. Data are presented as the mean ± SE. n.s., nonsignificant difference; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided).

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