Figure 5.

Sirt1 contributes to the resveratrol inhibitory effect of PMA-induced IkB phosphorylation. HBMEC were transiently transfected with a scrambled siRNA sequence (Mock) or a siRNA designed to downregulate Sirt1 (siSirt1) as described in the Methods section. (A) Mock and siSirt1-transfected HBMEC were then serum-starved for 30 minutes in the presence or not of 50 μM resveratrol, then treated with 1 μM PMA for the indicated time. Lysates were isolated, electrophoresed via SDS-PAGE and immunodetection of phosphorylated IκB (P-IκB) and GAPDH proteins was performed as described in the Methods section. (B) Total RNA isolation, RT-PCR, and qPCR were performed as described in the Methods section to assess Sirt1 gene expression in the Mock and siSirt1-transfected HBMEC. (C) Quantification was performed by scanning densitometry of the autoradiograms obtained in (A).

Notes: Data were expressed as the percent of basal phospho-IκB/GAPDH ratios in vehicle (open circles) and resveratrol pre-treated cells (closed circles). Densitometric data of a representative blot out of three is shown.