Effect of resveratrol on nicotinamide and/or antisense oligonucleotide-induced specific inhibition of Sirt-1 expression, Runx2 acetylation, Runx2 target genes (i.e.osteocalcin) or activation of PPAR-γ during osteoblastic differentiation of MSCs in monolayer culture.

<p>Cells were either untreated or treated with resveratrol (1 µM), nicotinamide (10 mM) or with Sirt-1 <i>antisense</i> (1 µM) or sense oligonucleotides (1 µM) in the presence of lipofectin alone or cells were pre-treated with resveratrol for 4 h followed by co-treatment with Sirt-1 antisense or sense oligonucleotides in the presence of lipofectin for 24 h or/and with nicotinamide over 21 days with osteogenic induction medium in monolayer cultures. (A) Whole cell lysates (500 ng/lane) were fractionated and subjected to western blotting with antibodies against Sirt-1 and β-actin. Synthesis of the housekeeping protein β-actin was unaffected. (B) Whole-cell extracts were prepared, immunoprecipitated with an anti-Runx2 antibody, and subjected to western blot analysis using an anti–acetyl-lysine antibody. The same blots were re-probed with an antibody to anti-Runx2. Whole cell lysates (500 ng/lane) were fractionated and analyzed by immunoblotting using anti-osteocalcin (C) or anti-PPAR-γ (D) antibodies and β-actin. Synthesis of the housekeeping protein β-actin was unaffected.