SIRT1 overexpression after transient transfection decreases senescence and restores cell proliferation. PT-ECFCs were untransfected (Mock), transiently transfected with pCMV-sport6 vector (Empty) or the pCMV-Sport6-SIRT1vector (SIRT1), and treated or not with NAM (1mM). (A) Transient overexpression of SIRT1 was monitored by western blot (upper panel) together with expression of p16^INK4a and p21^WAF. After 24 or 48 hours, cells were lysed and 30 µg of lysates were resolved on 4% to 12% SDS-PAGE gradient under reducing conditions. Blots were probed with anti-SIRT1,-p16 ^INK4a, -p21^WAF polyclonal antibodies or with anti–β-actin polyclonal antibody as a loading control. (B) Fold induction of SIRT1, p16 ^INK4a, and p21 ^WAF expression in SIRT1-transfected PT-ECFCs compared with cells transfected with the empty vector at the same time point. Bars represent mean ± SEM of the relative intensities of 8 independents experiments. *P < .05; **P < .01; *** P < .001. (C) Two days after transfection, SA-β-gal staining was performed for evaluation of the senescent status (left panels). A representative image of CT-ECFCs is shown. Other images correspond to PT-ECFCs after different treatments (original magnification ×20; scale bar, 100 µm). SA-β-gal–positive cells were counted and presented as percentage of senescent cells per total cell number (Right panel). Data are means ± SEM of 12 independents samples. For each, experiments were carried in triplicate. *P < .05; **P < .01. (D) Proliferation rate was determined by BrdU incorporation assay after transient transfection in 7500 ECFCs. Results were expressed as arbitrary units of spectrophotometric measurements. Data are means ± SEM of 12 independent samples. For each, experiments were performed in triplicate. *P < .05; **P < .01.