Piwil2 Activates STAT3 and Inhibits P53 Figure 7. A schematic model for the involvement of Piwil2 in anti-apoptosis in HeLa cells. 1, Piwil2 binds with monomeric STAT3 and recruited it to c-Src. 2, STAT3 is phosphorylated by c-Src and then dimerized. 3, dimeric pYSTAT3 translocates into the nucleus and bind to P53 promoter, repress P53 transcription and thus inhibit apoptosis. doi:10.1371/journal.pone.0030999.g007 Western Blot Analysis Harvested cells were lysed in ice-cold universal protein extraction buffer (Bioteke, Beijing) supplemented with protease inhibitor cocktail (Roche, USA) for 30 min. Cell lysates were separated on 4–12% SDS-page gel and transferred to a nitrocellulose membrane. Membranes were blocked in TBS-T Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween, PH 7.6) supplemented with 5% nonfat dry milk. The membranes were incubated overnight at 4uC with the indicated primary antibody: rabbit polyclonal anti-Piwil2 (Santa Cruz, USA), rabbit polyclonal anti-STAT3 (Abcam, UK), rabbit polyclone anti-pYSTAT3 (CST, USA), rabbit polyclonal anti-c-Src (Abcam, UK), rabbit polyclonal anti-pY416-c-Src (CST, USA), mouse monoclonal anti- GAPDH (Boster, China), mouse monoclonal anti-HA-tag (CST, USA), and rabbit polyclonal anti-Myc-tag (Santa Cruz, USA), followed by 5 minutes washes in TBS-T for three times and incubation with HRP-labeled secondary antibody (Zhongshan Table 1. Sequences of deployed siRNAs. siRNAs Piwil2 sense Piwil2 antisense c-Src sense c-Src antisense STAT3 sense STAT3 antisense P53 sense P53 antisense Sequences CUA UGA GAU UCC UCA ACU ACA GAA G [24] CUU CUG UAG UUG AGG AAU CUC AUA GUU [24] AAC AAG AGC AAG CCC AAG GAU dTdT AUC CUU GGG CUU GCU CUU GUU dTdT CAU CUG CCU AGA UCG GCU AdTdT UAG CCG AUC UAG GCA GAU GdTdT GAC UCC AGU GGU AAU CUA CdTdT GUA GAU UAC CAC UGG AGU CdTdT Nonspecific duplex siR-RiboTM Negative Control (Ribobio, China) doi:10.1371/journal.pone.0030999.t001 Goldenbridge, China) in TBS-T for 1 hour at RT. The membranes were detected with chemiluminescent HRP substrate (Millipore, USA). Immunofluorescence Transfected cells were fixed with 4% formaldehyde in PBS for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 1% BSA for 30 min, incubated overnight at 4uC with primary antibody and finally incubated with FITC-labeled secondary antibody (Zhongshan Goldenbridge, China) for 1 hour at RT. Each step was followed with 5 minutes washes in PBS twice. The prepared specimens were counterstained with 5 mg/ mlDAPI for 2 min and observed with a fluoresence microscope (Olympus, Japan). Immunoprecipitation Prepared cell lysates were incubated with 0.8 mg antibody against HA-tag or Myc-tag for 2 hours at 4uC with gentle inverting, then incubated with 20 ml of protein G&A agarose (Beyotime, China) overnight, and precipitated by centrifugation at 12,000 g for 1 min. Complexes were washed four times in ice-cold PBS Buffer (pH 7.4), and electrophoresed on 4–12% SDS-page gel. Western blot detection was carried out as described earlier. Chromatin immunoprecipitation (ChIP) assays and real- time PCR Preparation of chromatin-DNA and ChIP assays were per- formed as described by the manufacturer’s protocol of EZ-Zyme Chromatin Prep Kit (Millipore, USA) and Chromatin Immuno- precipitation Kit (Millipore, USA). Antibody against di-methylated H3K4 and di-methylated H3K9 were purchase from Abcam (UK). Purified DNA was subjected to PCR using primers specific for a 127-bp region spanning the STAT3-binding site in the P53 promoter. The sequences of the PCR primers used are as follows: PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e30999