We also predicted and then confirmed experimentally how the system responds to sequential thermal insults, or stepwise increases in temperature

n of proteins, whole cell lysates from cells treated with nicotinamide, resveratrol or combination of both of them were analyzed by western blotting using anti-acetyl lysine antibody. As shown in Fig. 6C, nicotinamide induced acetylation of several proteins, whereas resveratrol suppressed the acetylation 11885967” of these proteins. These findings suggest that resveratrol-activated Sirt-1 plays an important role in inhibiting nicotinamide-activated PPAR-c/ NCoR complex resulting in a decrease of Runx2 acetylation. examined whether the inhibitory effect of resveratrol on Runx2 acetylation is Sirt-1 dependent. The Sirt-1 specific oligonucleotidetransfected cells efficiently knocked down Sirt-1 protein levels during osteogenesis in vitro, and this abolished the ability of resveratrol to deacetylate Runx2 in resveratrol and/or nicotinamide-stimulated cells in monolayer cultures. Interestingly, the acetylation content of Runx2 was higher in cells treated with specific antisense oligonucleotides than in cells treated with or without sense oligonucleotides, suggesting the higher acetylated content of Runx2 protein is related to downregulated Sirt-1 expression and Runx2 could be a substrate for Sirt-1 deacetylase. To examine and establish a correlation between Sirt-1 and the activity of Runx2, western blot analysis with anti-osteocalcin antibody was performed. As shown in Fig. 8C, osteocalcin protein was downregulated in cells treated with nicotinamide and specific antisense oligonucleotides, compared to the cells treated with or without sense oligonucleotides. 17622149” Taken together, these results suggest that downregulation of Sirt-1 in MSCs can decrease Runx2 activities and its downstream target genes. To further investigate, if the acetylation of Runx2 by Sirt-1 downregulation, has an effect on the expression of PPAR-c protein, western blot analysis with anti-PPAR-c antibody was performed. As shown in Fig. 8C, PPAR-c protein was elevated in cells treated with specific antisense oligonucleotides, compared to the cells treated with or without sense oligonucleotides. These results suggest that downregulation of Sirt-1 in MSCs can increase adipogenic differentiation and expression of the adipose transcription Danoprevir manufacturer regulator PPAR-c and modulate the expression of downstream target genes. Discussion Specific antisense oligonucleotides downregulate Sirt-1 in MSCs in vitro To investigate whether specific antisense oligonucleotides against Sirt-1 inhibit Sirt-1 expression, MSCs were transfected with specific antisense or sense oligonucleotides derived from nucleotide sequence coding upstream part of catalytic domain of Sirt-1 protein. The immunofluorescence analysis as well as the immunoblot assays showed that the specific antisense oligonucleotides reduced the levels of Sirt-1 expression and nuclear localization. In contrast, the control sense oligonucleotide had no effect on Sirt-1 expression. The results indicated that treatment with Sirt-1 antisense oligonucleotides inhibited Sirt1 expression specifically and concentration dependently and the inhibition was not related to non-specific generegulatory events. Downregulation of Sirt-1 expression by antisense oligonucleotides enhances Runx2 acetylation, PPAR-c activation and inhibits expression of Runx2 target genes during osteogenic differentiation of MSCs in monolayer cultures Based on the results of co-immunoprecipitation assays, Sirt-1 interacts directly with Runx2 in vitro, which raises the possibility that Runx2 may