The consequence of p53 accumulation in hESCs.

<p>(A) Cell cycle analysis. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) and treated with RA were stained with PI and subjected to flow cytometry analysis to determine DNA content. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#s2" target="_blank">Results</a> quantitated as fold change in cell cycle are shown. (B) qRT-PCR. RNA from hESCs treated with RA for 4 d or Adr for 6 h were subjected to qRT-PCR assay using primers specific for human <i>CDKN1A</i>. (C) ChIP. p53-bound chromatin was immunoprecipitated from hESCs, and p53 enrichment on <i>CDKN1A</i> was analyzed by qRT-PCR using primers encompassing p53REs (*, <i>p</i><0.05). Scheme representing location of p53RE and primers used for ChIP-qRT-PCR are shown on the top (asterisk indicates the 3′ end of the gene). (D) hESCs treated with RA or Adr were lysed, and cell lysates were blotted for γ-H2AX. (E) Apoptosis assay. hESCs treated as in (D) were stained with Annexin V and PI, and percent apoptotic cells was determined by flow cytometry (mean ± SEM). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s004" target="_blank">S4</a>.).