Senescence of PT-ECFCs is associated with changes in expression for cell-cycle checkpoint factors and Rb phosphorylation. p53, p21WAF, and p16 INK4a expression and Rb phosphorylation were assessed as senescence markers. (A) Relative mRNA expression of different cell-cycle markers was determined by quantitative RT-PCR analysis on a Stratagen Mx3000. For each gene, data were normalized to the housekeeping gene RPL13A. Graphs are representative of 4 and 10 samples for CT and PT-ECFCs, respectively. Experiments were performed in duplicate. *P < .05. (B-D) The level of proteins involved in the regulation of the G1 phase was examined by immunoblot on whole-cell lysates from CT- and PT-ECFCs as described in “Patients, materials, and methods.” Representative experiment for p53 (B), p21WAF (C), and p16INK4a (D) in 4 independent CT and PT-ECFCs are shown in upper panel. A total of 30 µg of cell lysate was loaded on 4% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gradient under reducing conditions. Blots were probed with anti-p53, -p16 INK4a, -p21WAF polyclonal antibodies or with anti–β-actin polyclonal antibody as loading control. Protein quantification was presented by bar graphs (lower panels) showing means ± SEM of densitometry ratio for each protein normalized to β-actin of 4 independent immunoblots. *P < .05; **P < .01. (E) Changes in the phosphorylation state of Rb were analyzed by immunoblots using antibody against total (Rb) and phosphorylated Rb (P-Rb). Representative experiment is shown. Histograms display normalized staining intensity measured by densitometry. Left, P-Rb to β-actin ratio; middle, Rb to β-actin ratio; and right, P-Rb to Rb ratio. *P < .05
