Fig. 4. PPAR binds to the PPRE-containing region in the p16 gene promoter. (A) Schematic diagram of the p16 gene promoter. `PPRE' denotes putative PPAR-binding sites and `control' denotes a region located immediately downstream of PPRE. The numbers are the positions upstream of the p16 gene translation initiation site. (B) Soluble chromatin was prepared from 2BS cells [young (Y; PD25) or senescent (S; PD62)] or WI-38 cells [young (Y; PD20) or senescent (S; PD50)] treated (+) or not (–) with 20 µM troglitazone for 48 hours. (C-E) Soluble chromatin was prepared from young or senescent 2BS cells treated or not with 20 µM troglitazone (C) or 10 µM pioglitazone (D,E) for 48 hours. Precipitated DNA samples were amplified with primers recognizing the PPRE domain. ChIP assays were quantified by real-time PCR (C,E). Values are expressed relative to the controls (untreated young 2BS cells), which were set as 1. Values are the mean ± s.d. of triplicate points from a representative experiment (n=3), which was repeated three times with similar results. ^*P0.05. (F) Soluble chromatin was prepared from young or senescent 2BS cells treated or not with 10 µM GW9662 for 48 hours. Precipitated DNA samples were amplified with primers recognizing the PPRE domain. (G) Soluble chromatin was prepared from young or senescent 2BS cells treated or not with 20 µM troglitazone for 48 hours. Precipitated DNA samples were amplified with primers recognizing the control element as indicated in A. IP was performed with an antibody against PPAR and DNA was amplified using primer pairs as indicated. As negative controls, the no antibody (No Ab) sample is an immunoprecipitation that did not contain antibody, and antibody against β-actin was used as an irrelevant antibody control. The input sample (Input) contained 0.5% of the total starting chromatin. The ChIP assays were repeated three times, and results of a representative experiment are shown.