RSV reverts senescence by increasing SIRT1 expression. PT-ECFCs were treated with solvent alone (DMSO) or 1µM RSV for different times. (A) Time-dependent changes in the level of SIRT1 were determined by quantitative RT-PCR analysis on a Stratagen MX3000. Changes in SIRT1 mRNA were normalized to the housekeeping gene RPL13A, as described in “Patients, materials, and methods.” Results were expressed as fold induction of SIRT1 mRNA level in RSV-stimulated PT-ECFCs compared with DMSO-treated PT-ECFCs in 10 independent samples. Data are means ± SEM, *P < .05; **P < .01. (B) RSV- and DMSO-treated PT-ECFCs were analyzed by chromatin immunoprecipitation assays. Relative enrichment of 2 chromatin marks, H3K4me3 (active chromatin) and H3K9me3 (repressed chromatin) at the SIRT1 promoter was determined in PT-ECFCs by quantitative PCR after 48 hours of treatment and calculated as the ratio between bound sample and the input DNA (total DNA after crosslinking). Data are means ± SEM of 6 independent samples. Experiments were performed in triplicate. **P < .01. IgG, immunoglobulin G. (C) Time-dependent modulation in SIRT1, p16^INK4a, and p21 ^WAF proteins in RSV-treated PT-ECFCs. Each whole-cell lysate (30 µg) was 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. Staining intensity was measured by densitometry and normalized to β-actin. Results were expressed as fold induction of protein expression in RSV-stimulated PT-ECFCs compared with DMSO-treated PT-ECFCs. Bars represent mean ± SEM of the relative intensities in 12 independents samples. *P < .05; **P < .01; ***P < .001. (D) Immunofluorescence staining of RSV-treated or not PT-EFCFs after 48 hours for SIRT1 (green, left panels). Nuclei were counterstained with DAPI (blue). Representative images are shown from the 2 groups (original magnification ×20; scale bar, 50 µm). White arrows indicate low SIRT1 staining. Bars represent means ± SEM of the number of cells with SIRT1 nuclear staining relative to the total cell number.* P < .05. (E) After 48 hours of DMSO or RSV treatment, in the presence or absence of 1mM NAM, SA-β-gal staining of PT-ECFCs was performed for evaluation of the senescence status. The SA-β-gal–positive cells were counted and presented as percentage of the total cells. Data are means ± SEM of 10 independents samples. For each, experiments were carried in triplicate.*P < .05; ***P < .001.