STAT3 Signaling in Pancreatic Cancer Figure 4. Inhibition of STAT3 enhances the inhibitory effects of EGCG on motility and cell viability of pancreatic cancer cells. (A), AsPC-1 and PANC-1 were transfected with STAT3 shRNA. The expression of STAT3 was performed by Western blotting. (B), AsPC-1 scratch assay. AsPC-1 scrambled and STAT3 shRNA cells were cultured in 6 well dishes. The scratch was marked when the dishes were 50% confluent. Pictures were taken after the cells were treated with EGCG and incubated for 0, 24 and 48 h. (C), Cell viability assay. AsPC-1 and PANC-1 (scrambled and STAT3 shRNA) cells were seeded and treated with EGCG (0, 20, 40, 60 mM). After 72 h of treatment, cell viability was performed by XTT assay. Data represent mean 6 SD. * or ** = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0031067.g004 primers were purchased from Realtimeprimers.com. All reactions were performed in triplicate, and the relative expression of target mRNA in each sample was normalized with that of mean GAPDH. PCR primers sequences: GAPDH, forward primer 59- GAG TCA ACG GAT TTG GTC GT -39; reverse primer 59- TTG ATT TTG GAG GGA TCT CG -39; STAT3, forward primer 59- CCT TTG ACA TGG AGT TGA CC -39; reverse primer 59- TAA AAG TGC CCA GAT TGC TC -39; Cyclin D1, forward primer 59- TTC AAA TGT GTG CAG AAG GA -39, reverse primer 59- GGG ATG GTC TCC TTC ATC TT -39; c-Myc, forward primer 59- CGA CGA GAC CTT CAT CAA AA -39, reverse primer 59- TGC TGT CGT TGA GAG GGT AG -39; Survivin, forward primer 59- TCC CTG GCT CCT CTA CTG TT -39, reverse primer 59- TGT CTC CTC ATC CAC CTG AA -39; VEGF, forward primer 59- AGA CAC ACC CAC CCA CAT AC -39, reverse primer 59- TGC CAG AGT CTC TCA TCT CC -39; BclXL forward primer 59- GCT CTC ACT CCC AGT CCA AA - 39, reverse primer 59-GCT GAG GCC ATA AAC AGC TC -39. Immunofluorescent staining AsPC-1 and PANC-1 cells were cultured in RPMI medium containing 10% FBS and treated with EGCG (0, 40, 60 mM) for 24 h. Cells were then fixed with 4% paraformaldehyde and stained with antibodies against STAT3 (mouse monoclonal IgG1; Cell Signaling) at 4uC overnight. Cells were washed and again incubated with anti-mouse-FITC secondary antibody (Sigma) along with DAPI (0.5 mg/ml). Stained slides were mounted with mounting medium and visualized under a fluorescence micro- scope. For better visuality, the color of DAPI was changed from blue to red. The green color represents the expression of STAT3. Western blotting analysis To detect different proteins, AsPC-1 and PANC-1 cells treated with EGCG (0–60 mM) were washed with PBS and lysed in RIPA buffer containing 16 protease inhibitor cocktail. The lysates were centrifuged and the supernatant was collected. Protein concentra- tions were determined using the Bio-Rad Protein Assay (Bio-Rad). Protein extracts (40 mg) were separated on 12.5% SDS-PAGE. Transferred membranes were blocked using 5% nonfat dry milk and incubated overnight with primary antibodies at 1:1,000 dilutions in TBS, followed by secondary antibodies conjugated with horseradish peroxidase at 1:5,000 dilutions in TBS-Tween 20 for 1 hour at room temperature. Membranes were developed using ECL Substrate. Protein bands were visualized on X-ray film using an enhanced chemiluminescence system. PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31067