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

Figure 5

HIF-1α expression. BM-MSCs were cultured for 48 h in normoxic or in hypoxic conditions in the absence (vehicle) or in presence of 2 μM S1PR1 receptor antagonist, W146, or 2 μM S1PR1 receptor agonist, SEW2871. (a) Representative immunofluorescence confocal images of cells cultured on glass coverslips in the indicated experimental conditions, fixed and immunostained with antibodies against HIF-1α (green). Scale bar 50 μm. The images are representative of at least three independent experiments with similar results. (b) Densitometric analysis of the intensity of the HIF-1α fluorescence signal performed on digitized images. Data are mean ± S.E.M. (c) Western blotting analysis of HIF-1α. Cell lysate (30 μg) obtained from BM-MSCs cultured in hypoxia and treated as reported above was loaded onto SDS-PAGE and protein immunodetected by a specific antibody. β-Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments is shown in the graph (mean ± S.E.M.). Significance of differences in (b) (one-way ANOVA and Newman-Keuls multiple comparison test), versus normoxia, versus vehicle hypoxia; in (c) (Student’s t-test), versus vehicle.

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