Analysis of Polycomb EZH2, M33 and BMI1 binding at the <i>INK4a/ARF</i> region.

<p>A) qPCR analysis of p16^INK4a and p19^ARF in young (P3), senescent (P10) and in <i>Bmi1</i> and <i>M33</i> mutant MEFs. B) B-galactosidase staining to detect senescent cells at passage 3 (P3) and passage 10 (P10). C) qPCR analysis of the mRNA levels of Polycomb EZH2 and Bmi1 in the indicated cells. D–F) Schematic diagram of the <i>INK4a/ARF</i> locus: amplified regions that were tested in ChIP experiments are indicated by red bars (sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005622#pone-0005622-t001" target="_blank">Table 1</a>). Wild type P3 (young), P10–12 (senescent), M33−/− (P4) and Bmi−/− (P4) MEFs were subjected to ChIP assays using anti EZH2, BMI1 and M33 antibodies. DNA enrichment was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005622#s2" target="_blank">Materials and Methods</a>. Bars represent the mean+/−s.d. of quantifications from two to four separate immunoprecipitations analyzed in triplicate.