Obesity-Induced Methylation of Osteopontin Contributes to Adipogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells

Figure 4

OPN retards the adipogenic differentiation of ASCs mainly in an extracellular manner. (a) WT and OPN^−/− ASCs were cultured in adipogenic differentiation medium supplemented with OPN-specific antibody (anti-OPN Ab) or isotype control (each 10 μg/ml), then stained with Oil Red O after 4 days. (b) OPN^−/− and WT ASCs, transfected with secreted OPN (sOPN), intracellular OPN (iOPN), or pcDNA3.0, were cultured in adipogenic differentiation medium for 6 days, then stained with Oil Red O. (c) OPN^−/− and WT ASCs were cultured in adipogenic differentiation medium supplemented with recombinant OPN (rmOPN, 1 μg/ml) or PBS [27] for 4 days and stained with Oil Red O. (d) hASCs, from overweight (25 ≤ BMI< 30) and normal (BMI < 25) human subjects, were cultured in adipogenic differentiation medium supplemented with OPN-specific antibody (anti-OPN Ab) or isotype control (each 10 μg/ml) for 6 days, then stained with Oil Red O. (e) Overweight and normal ASCs were cultured in adipogenic differentiation medium exposed to PBS and recombinant OPN protein (each 1 μg/ml) (rOPN) for 4 days, and then stained with Oil Red O. (f) Overweight and normal ASCs, transfected with secreted OPN (sOPN), intracellular OPN (iOPN), or pcDNA3.0, were subjected to adipogenic differentiation medium for 6 days, then stained with Oil Red O. (g) WT and OPN^−/− ASCs supplemented with blocking antibodies against integrin αν/β1 or CD44 or an isotype control (lgG; each 5 μg/ml) was cultured in adipogenic differentiation medium for 4 days and stained with Oil Red O. Adipocyte size was quantification analyzed by an imaging system (ImageJ, National Institutes of Health). (Bar = 50 μm; adipocytes counted in four random fields from two wells per group.) Values are means ± SEM. ; ; . All experiments were repeated at least thrice.

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