FIG. 3.
Soluble factor induced changes in the strength and distribution of cell adhesion molecules (CAMs). (A) Confocal images of soluble-factor-treated MSCs stained with Phalloidin (F-actin, red), anti-vinculin (green), and DAPI (nucleus, blue) (scale bar=20 μm). Magnified images of representative vinculin structures are shown in insets. (B) Centrifuge-based adhesion assay was used to determine the effects of soluble factor treatment on the adhesion of MSCs on tissue culture plastic coated with collagen—10 μg/ml (COL) or fibronectin—10 μg/mL (FBN). TGF-β1 treatment resulted in an increased fraction of adherent cells. Results are reported as average±SEM (n=8). (C) Histograms from flow cytometry were analyzed using FACS-DIVA for mean fluorescence intensity (MFI). Surface integrin expression of PDGF-treated cells was unaffected and vascular cell adhesion molecule-1 (VCAM-1) expression was slightly decreased, whereas TGF-β1 individually and in combination increased both integrin expression and reduced VCAM expression significantly compared with the control. Results are reported as average±SEM (n=3). (D) Histograms from flow cytometric analysis of surface CAMs using fluorescent-labeled antibodies for αv (PE), β1 (FITC) integrins, and VCAM-1 (APC) on MSCs after 24 h treatment with soluble factors. Gated percent positive population of MSCs compared with the negative population (black histogram) are indicated as mean±SEM on top right of overlayed histograms (red for CM, green for PDGF, blue for TGF-β1, and violet for PDGF+TGF-β1). Statistical significances are indicated using (*) for p<0.05, (**) for p<0.005, and (***) for p<0.0005. Color images available online at www.liebertpub.com/scd