Figure 9.
Laminin-mediated binding of PC3-derived sEVs induces endothelial morphogenesis in HUVEC. (A) Western blot analysis of laminin γ1 and total laminin levels in laminin γ1 KD HUVEC. (B) Fluorescence images of WT or laminin γ1 KD HUVECs expressing GFP and sEVs-PC3-CD63Halo7-TMR bound to the cells, observed by confocal microscopy. Cells were fixed after 1-h incubation with sEVs. Since not all cells necessarily express GFP, many fluorescent spots of sEVs were observed either in regions containing non-expressing cells or potentially on the glass surface. Nevertheless, the number of sEVs bound to both the apical and basal surfaces of the cell PM can be quantified, as shown in Fig. 4. (C) Quantification of sEV particles bound to both apical and basal PM of WT or laminin γ1 KD HUVEC by confocal microscopy (n = 15 cells). (D) Time course analysis of the number of sEV particles bound to the basal PM of live WT or laminin γ1 KD HUVEC, monitored using TIRFM (n = 12 cells). (E) Fluorescence images of HUVEC-expressing GFP after 12 h of treatment with PC3-derived sEVs or 10 ng/ml VEGF. Yellow arrowheads show branched protrusions of HUVEC. (F) WT (+)sEV image from E showing protrusion lengths measured along yellow lines as indicated. (G) Average protrusion lengths across all examined cells before and after treatment with sEVs or VEGF. (H) Changes in average protrusion length after treatment with sEVs or VEGF, relative to untreated conditions (n = 16 cells). (I) Average total protrusion length per cell before and after treatment with sEVs or VEGF. (J) Variations in average total protrusion length per cell after treatment with sEVs or VEGF, relative to untreated conditions (n = 16 cells). Data are presented as mean ± SE. n.s., nonsignificant difference; *P < 0.05; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided). Source data are available for this figure: SourceData F9.

Laminin-mediated binding of PC3-derived sEVs induces endothelial morphogenesis in HUVEC. (A) Western blot analysis of laminin γ1 and total laminin levels in laminin γ1 KD HUVEC. (B) Fluorescence images of WT or laminin γ1 KD HUVECs expressing GFP and sEVs-PC3-CD63Halo7-TMR bound to the cells, observed by confocal microscopy. Cells were fixed after 1-h incubation with sEVs. Since not all cells necessarily express GFP, many fluorescent spots of sEVs were observed either in regions containing non-expressing cells or potentially on the glass surface. Nevertheless, the number of sEVs bound to both the apical and basal surfaces of the cell PM can be quantified, as shown in Fig. 4. (C) Quantification of sEV particles bound to both apical and basal PM of WT or laminin γ1 KD HUVEC by confocal microscopy (n = 15 cells). (D) Time course analysis of the number of sEV particles bound to the basal PM of live WT or laminin γ1 KD HUVEC, monitored using TIRFM (n = 12 cells). (E) Fluorescence images of HUVEC-expressing GFP after 12 h of treatment with PC3-derived sEVs or 10 ng/ml VEGF. Yellow arrowheads show branched protrusions of HUVEC. (F) WT (+)sEV image from E showing protrusion lengths measured along yellow lines as indicated. (G) Average protrusion lengths across all examined cells before and after treatment with sEVs or VEGF. (H) Changes in average protrusion length after treatment with sEVs or VEGF, relative to untreated conditions (n = 16 cells). (I) Average total protrusion length per cell before and after treatment with sEVs or VEGF. (J) Variations in average total protrusion length per cell after treatment with sEVs or VEGF, relative to untreated conditions (n = 16 cells). Data are presented as mean ± SE. n.s., nonsignificant difference; *P < 0.05; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided). Source data are available for this figure: SourceData F9.

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