Piwil2 Activates STAT3 and Inhibits P53 Figure 6. The specificity and domains of Piwil2-Stat3 interaction. A, schematic of different Piwil2 deletion constructs. B, Piwil2 deletion mutants with HA-tag were respectively co-transfected in HeLa cells with Myc-tagged STAT3, coIP assay revealed that mutants with PAZ domain deletion (pPAZ and pP+P) failed to bind with STAT3. C, Western blot analysis showed that PAZ deletion mutants failed to induce phosphorylation of STAT3. D, Apoptosis analysis showed that PAZ deletion mutants failed to inhibit apoptosis in transfected HeLa cells. doi:10.1371/journal.pone.0030999.g006 Materials and Methods Plasmids and siRNAs The coding sequences of Piwil2, STAT3 and c-Src were amplified by RT-PCR and cloned into the vector pcDNA3.1 (Invitrogen, USA) with an upstreaming Myc- or HA-tag respectively. TRIpure reagent, a gel extraction kit, and a high purity plasmid preparation kit were purchased from Bioteke Corporation (Beijing, China) for total RNA extraction, PCR product purification, and plasmid preparation. The specific siRNAs (described in Table 1) and universal negative control dicer substrate duplex were synthesized and purchased from RiboBio Co. Ltd (Guangzhou, China). The putative STAT3 binding site in p53 gene promoter was obtained and mutated through a PCR method, then cloned into a pVAX-Luciferase plasmid (maintained in our laboratory). The sequences of the PCR primers used are as follows: wildtype forward: 59- CGC GGA TCC AGC TCT GGC TTG CAG AAT TTT CCA C -39; mutated forward: 59- CGC GGA TCC AGC TCT GGC GGG CAG AAT TTT CCA C -39; universal reverse: 59- CCG GAA TTC CCG GAG GAA GCA AAG GAA ATG G -39. Cell culture Cervical cancer cell line HeLa was maintained in our laboratory [34]; hepatocellular carcinoma cell line HepG2 was obtained from State Key Laboratory of Biotherapy and Cancer Centre of West China Hospital [35]. Both cell lines were maintained in RPMI- 1640 medium (Gibco, USA) containing 10% heat-inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, on 25 cm2 culture dishes in a humidified atmosphere containing 5% CO2 incubator at 37uC. The cells were passaged by trypsinization every 2–3 days. The transfection was performed with lipofectamine 2000 solution (Invitrogen, USA) according to the manufacturer’s protocol and transfected cells were harvested and analyzed 24 hours after transfection, unless stated otherwise. Empty plasmid DNA was applied to transfection as negative control. And in some experiments, stable overexpression transfec- tants selected by being cultured in medium containing 1 mg/ml of G418 (Solarbio, China) for one month were also examined. To inhibit tyrosine kinase activity, HeLa cells were pretreated with 25 mM AG490 (Sigma-Aldrich, USA) for 24 hours or 10 mM PP2 (Sigma-Aldrich, USA) for 30 minutes. All following experiments were repeated at least three times unless stated otherwise. Apoptosis assay Apoptotic rates were analyzed by a COULTER EPICS XL flow cytometer (Beckman, USA) using an Annexin V-EGFP Apoptosis Detection Kit (Bestbio, Shanghai). Annexin V/PI staining and fluorescence intensity measurements were performed according to the manufacturer’s instruction. PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30999