Fig. 2.

A: reporter gene assay demonstrates the knockdown effects of NPM [by small interfering RNA (siRNA)] on TNF-α (10 ng/ml)-induced NF-κB reporter activity in cultured primary human coronary arterial endothelial cells (HCAECs). The resulting overexpression of NPM with respect to endothelial NF-κB activation is also shown, as well as effects of resveratrol (10 μmol/l) on TNF-α (10 ng/ml)-induced NF-κB reporter activity. Cells were transiently cotransfected with NF-κB-driven firefly luciferase and CMV-driven Renilla luciferase constructs, followed by NPM knockdown, NPM overexpression, or TNF-α stimulation. Cells were then lysed and subjected to luciferase activity assay. Following normalization, relative luciferase activity was obtained from 6 independent transfections. Data are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α only; &P < 0.05 vs. NPM overexpression. B: effect of knockdown of SIRT-1 (siRNA) and/or resveratrol (10 μmol/l) treatment on mRNA expression of NPM in cultured HCAECs. Analysis of mRNA expression was performed by real-time QRT-PCR. β-Actin was used for normalization. Data are means ± SE (n = 5 for each group). C: NPM mRNA expression in carotid arteries of ad libitum-fed young (3 mo old), ad libitum fed aged (28 mo old), and caloric-restricted aged (28 mo old) F344 rats. Analysis of mRNA expression was performed by real-time QRT-PCR. β-Actin was used for normalization. Data are means ± SE (n = 5 for each group). *P < 0.05. vs. young; #P < 0.05 vs. ad libitum-fed aged. All animal use protocols were approved by the Institutional Animal Care and Use Committee of the New York Medical College, Valhalla, NY.