Figure 3: Fibronectin and laminin mediate MV-promoted trophoblast migration.

(a) Mass spectrometry was performed on MVs isolated from ES cells. The 10 most abundant proteins identified are listed. Arrows highlight proteins predicted to most likely mediate the migration-promoting effects of MVs. (b) Lysates of ES cells (WCL), and the MVs that they generated (MVs), were immunoblotted for fibronectin, laminin α5, the MV marker flotillin-2, the cytosolic-specific protein FAK, and β-actin, as a loading control. (c,d) Serum-starved HTR8/SVneo trophoblasts were treated with PBS (Vehicle) or with either (c) the fibronectin inhibitory peptide RGD or the laminin inhibitory peptide YIGSR or (d) a combination of the two inhibitors. The cells were then stimulated with ES cell MVs for ∼30 min before being lysed and immunoblotted for phosphorylated and total FAK and JNK. The ratio of each phospho-protein/total protein pair examined was determined and included on the blots. Note only the combination of RGD and YIGSR blocked integrin-mediating phosphorylation of FAK and JNK. (e) Images were taken of wound closure assays performed on HTR8/SVneo cells cultured in serum-free medium supplemented with the vehicle (top panels) or the RGD- and YIGSR-inhibitory peptides (bottom panels). Each culturing condition was further treated without (Serum free medium) or with either MVs from ES cells (ES cell MVs) or 1% serum. The dashed line indicates the width of the original wound. Scale bar, 250 μm. (f) The assays in e were quantified and plotted as the relative area of open wound. All values shown are presented as mean±s.e.m. (n≥3 independent experiments for each assay). Differences were analysed using Student’s t-test; *P<0.05, **P<0.01.