Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation
Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μM exogenous sphingosine-1-phosphate (exoS1P), 2 μM S1PR1 receptor antagonist, W146, or 2 μM S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μm. The images are representative of at least three independent experiments with similar results. Histogram represents quantitative analysis of Ki67 positive BM-MSC cell nuclei expressed as percentage of the total nuclei number. Data are mean ± S.E.M. (b) Cell proliferation analysis by cell counting. Synchronized BM-MSCs were collected and counted as reported in Section 2. Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μg) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. 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 are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): versus vehicle.