Fig. 7: sEV binding triggered intracellular Ca2+ signaling.
a Typical confocal microscopy images of Fluo-8H-loaded PZ-HPV-7 cells. sEVs (top) or HBSS (bottom) were added to the cells 2.5 min after the initial observation. Ionomycin was added at 20 min. Scale bar: 20 μm. b Time course of the fluorescence intensity of Fluo-8H in (a). c Normalized Ca2+ concentration after the addition of HBSS, sEVs isolated by ultracentrifugation, sEVs isolated using Tim-4 beads, and sEV-depleted culture medium (exomeres). d Normalized Ca2+ concentration after the addition of sEVs (5.0 × 109 particles/mL at final concentration) (closed diamond) derived from three donor cell types to three recipient cell types (9 combinations). sEV(−) indicates the results obtained after the exomeres were added (open diamond). e Normalized Ca2+ concentration of PZ-HPV-7 cells in which talin-1 activity was inhibited by expressing a dominant negative mutant (TalinDN) or treating the cells with inhibitors of IP3 receptor (IP3R) (2-APB), phospholipase C (PLC) (U73122), Src family kinase (SFK) (PP2), a PLC control reagent (U73343), or an SFK control reagent (PP3) after the addition of PC-3 cell-derived sEVs. sEV(-) indicates the results obtained after the exomeres were added. f Typical single molecules of mGFP-PLCγ2 (indicated by yellow arrowheads) were frequently recruited to the PZ-HPV-7 cell PM within 90 s after the addition of PC-3 cell-derived sEVs. The images were obtained by TIRF microscopy. g Number of mGFP-PLCγ2 spots recruited to the PM before and after the sEVs were added in the absence or presence of PP2/PP3. h Colocalization analysis of single sEV particles with single molecules of membrane-associated focal adhesion kinase (FAK), PLCγ1, and PLCγ2 in the cell PM. i Typical image sequence (every 30 ms) of dSTORM images of SF650B-Halo7-FAK domains (magenta) and single molecules of mGFP-PLCγ2 (green). Yellow arrowheads indicate colocalization events. j PLCγ2 molecules were frequently recruited to FAK-enriched domains. Density analysis of single molecules of mGFP-PLCγ2 on the SF650B-Halo7-FAK domains. In Fig. 7, “N” indicates the number of examined cells; data were presented as the means ± SE. Statistical analyses were conducted as described in “Statistics and reproducibility” in the Methods section.
