Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

Figure 3

Cytoskeleton organization and cortactin expression. BM-MSCs were cultured for 48 h in the absence (vehicle) or in presence of the following compounds: 1 μM exogenous sphingosine-1-phosphate (exoS1P), 2 μM S1PR1 receptor antagonist, W146, and 2 μM S1PR1 receptor agonist, SEW2871 and/or MMP-2/9 inhibitor, SB-3CT (5 μM or 10 μM). (a–h) Representative immunofluorescence confocal images of cells cultured on glass coverslips in the indicated experimental conditions, fixed and stained with Alexa 568-phalloidin to visualize actin filaments (red) and immunostained with antibodies against cortactin (green). Scale bar 50 μm. Arrows indicate filopodia and arrowheads indicate lamellipodia (L). (A–D) Magnifications of the indicated squared regions of interest showing the red and green fluorescence signals separately and together. Yellow-orange colour indicates colocalization between the two fluorescence signals. Scale bar 12 μm. The images are representative of at least three independent experiments with similar results. (i) Densitometric analysis of the intensity of the cortactin fluorescence signal performed on digitized images. Data are mean ± S.E.M. Significance of differences (one-way ANOVA and Newman-Keuls multiple comparison test): versus vehicle, versus SEW2871.

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