Accelerated senescence in PT-ECFCs is correlated to decreased SIRT1 levels associated to reducing activity. SIRT1 expression was assessed in ECFCs from CT and PT neonates at the RNA and protein level. (A) Relative SIRT1 expression was determined by quantitative RT-PCR analysis on a Stratagen Mx3000. Data were normalized to the housekeeping gene RPL13A. Graph is representative of 7 and 12 experiments performed in duplicated for CT- and PT-ECFCs, respectively. **P < .01. (B) Western blot analysis of SIRT1 from ECFCs of 4 representative CT and PT neonates was shown. Histograms display mean ± SEM of SIRT1 intensity measured by densitometry normalized to β-actin for 4 independent experiments. **P < .01. (C) Costaining between SA-β-gal activity and SIRT1 protein were realized as described in “Patients, materials, and methods.” CT- and PT-EFCFs were stained by immunofluorescence for SIRT1 protein (green). Nuclei were counterstained with 4,6 diamidino-2-phenylindole (DAPI) (blue). Representative images from the 2 groups are shown (original magnification ×20; scale bar, 50µm). Bars represent means ± SEM of the number of cells with a SIRT1 nuclear signal relative to the total cell number. **P < .01. (D) SA-β-gal activity negatively correlated with SIRT1 protein level in samples from all groups (n = 10, P3); r2, correlation coefficient of determination; r, Pearson correlation coefficient. (E) Changes in the acetylation state of p53 were analyzed by immunoblots using antibody against total and acetyl p53. Representative experiments is shown. Bars display densitometry analysis after normalization to β-actin as mean ± SEM of the relative intensities of 4 independent experiments. *P < .05. (F) Western blot analysis of SIRT1 from MNC of 4 representative CT and PT neonates was shown. Bars display densitometry analysis of SIRT1 after normalization to β-tubulin as mean ± SEM of the relative intensities of 2 independent experiments. *P < .05.