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

Figure 5

C/EBPs are involved in the OPN-directed adipogenic differentiation of ASCs. (a) Total RNA from CD or HFD ASCs was assayed for C/EBPα and C/EBPβ mRNA by real-time polymerase chain reaction (PCR). (b) Total RNA from WT or OPN^−/− ASCs was assayed for C/EBPα and C/EBPβ mRNA by real-time PCR. Total protein from OPN^−/− or WT ASCs was analyzed for OPN, C/EBPα, C/EBPβ (b), and p-AKT (g) by western blotting. (c) Total RNA and protein preps from the WT ASCs transfected with OPN siRNA (siOPN) or control siRNA (siNC) assayed for C/EBPα and C/EBPβ 3 days after transfection. (d) Total RNA and protein preps from OPN^−/− ASCs with rOPN (rOPN) or PBS were assayed for C/EBPα and C/EBPβ by real-time PCR and western blot, and (h) protein preps from the same cells were assayed for p-AKT by western blot 2 days after addition. WT ASCs were supplemented with OPN-specific antibody (anti-OPN Ab) (10 μg/ml) for 2 days. Total protein preps from WT ASCs with anti-OPN Ab (anti-OPN Ab) and control cells (lgG) were assayed for C/EBPα and C/EBPβ (e) by western blot. (f) Wild-type ASCs were supplemented with blocking antibodies against CD44 or integrin αν/β1 or an isotype control (each 5 μg/ml) for 2 days. Total RNA and protein preps from WT ASCs with CD44 (anti-CD44) or integrin αν/β1 Ab (anti-β1 Ab) and control cells (anti-lgG) were assayed for C/EBPα, C/EBPβ by real-time PCR and western blot. Values are means ± SEM. ; ; . All experiments were repeated at least thrice.

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