Piwil2 Activates STAT3 and Inhibits P53 Figure 3. Piwil2 phosphorylated STAT3 binds to P53 promoter. A, a potential STAT3 binding site in P53 promoter, 179 bp upstream of the first exon of P53. B&C, ChIP assays of chromatin prepared from transfected HeLa cells using the indicated antibodies followed by real-time PCR, which amplified a 127-bp region spanning the Stat3-binding site described above. B, STAT3 binding activity to P53 promoter showed significant changes 12 hours after transfection of pcDNA3.1-Piwil2 or siRNA. C, histone methylation level of P53 promoter was examined with anti-di- methylation H3K4 (darker) and anti-di-methylation H3K9 (lighter) antibody, respectively. D, Piwil2 suppressed the translation of P53 by regulating its promoter. HeLa cells were transfected with the indicated constructs. Relative luciferase activity is the ratio between firefly luciferase and renilla luciferase, adjusted to 100%. doi:10.1371/journal.pone.0030999.g003 Figure 4. c-Src involved in Piwil2-induced STAT3 phosphorylation. HeLa cells pretreated with JAK-inhibitor AG490 or Src-inhibitor PP2 were transfected with same amount of empty plasmid (+pcDNA3.1), pcDNA3.1-Piwil2 (+Piwil2) or Piwil2-specific siRNA (+siRNA), harvested and examined by western blot analysis. Non-pretreated cells were employed as negative control (NC). The results showed that PP2 rather than AG490 can deplete the activity of c-Src and block the regulation to STAT3 phosphorylation and P53 expression by Piwil2. doi:10.1371/journal.pone.0030999.g004 PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30999