STAT3 Signaling in Pancreatic Cancer Figure 2. EGCG inhibits JAK3/STAT3 pathway in pancreatic cancer. (A), AsPC-1 and PANC-1 cells were treated with EGCG (0–60 mM) for 48 h, and the expression of STAT3 was measured by q-RT-PCR. Data represent mean 6 SD. * or ** = significantly different from respective controls, P,0.05. (B), AsPC-1 and PANC-1 cells were treated with EGCG (0–60 mM) for 48 h. The expression of STAT3, p-STAT3, JAK3 and p-JAK3 was measured by Western blot analysis. b-actin was used as a loading control. (C), Expression of STAT3 in AsPC-1 and PANC-1 cells. Cells were treated with EGCG (0– 60 mM) for 48 h. After incubation, the expression of STAT3 was measured by immunoflurescence. DAPI was used to stain nuclei. For better visuality, the color of DAPI was changed from blue to red. The green color represents the expression of STAT3. Red color = nuclei. doi:10.1371/journal.pone.0031067.g002 cells enhances the inhibitory effects of EGCG on cell migration and motility. Our results demonstrate that activation of the STAT3 signaling pathway is critical for the growth of pancreatic cancer cells and suggest that EGCG targeting STAT3 signaling may be a potential therapeutic intervention for pancreatic cancer. Furthermore, the combination of EGCG with gemcitabine or CP690550 had additive/synergistic effects on cell viability and apoptosis. Materials and Methods Cell lines and culture conditions Human pancreatic cancer cell lines AsPC-1 and PANC-1 were purchased from the American Type Culture Collection (Manassas, VA), and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Thermo Scientific) and 1% antibiotic- antimycotic (Invitrogen) at 37uC in a humidified atmosphere of 95% air and 5% CO2. Cell transfection STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).The accession number was obtained from the Gene bank. The sequences of STAT3 shRNAs (accession number: NM_139276) are corresponding to the coding regions 398–416 (59- CCA CTT TGG TGT TTC ATA A-39), 1070–1088 (59-CCCGTCAACAAATTAAGAA-39), 1448–1466 (59-GCC TCT CTG CAG AAT TCA A-39) and 1935–1953 (59-GGA CAA TAT CAT TGA CCT T-39) nucleotides. AsPC-1 and PANC-1 cells were transfected with a mixture of shRNAs using Lipofetamine 2000 (Invitrogen). After 24 h of transfection, cells were treated with EGCG. Cells were used for cell viability detection, scratch assay, qRT-PCR and western blotting. XTT Assay Cells (16104 in 200 ml culture medium per well) were seeded in 96-well plate (flat bottom), treated with or without drugs and incubated for various time points at 37uC and 5% CO2. Before the end of the experiment, 50 ml XTT labeling mixture (final concentration, 125 mM XTT (sodium 2,3-Bis(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) and 25 mM PMS (phenazine methosulphate) per well was added and plates were incubated for further 4 h at 37uC and 5% CO2. The spectrophotometric absorbance of the sample was measured using a microtitre plate (ELISA) reader. The wavelength to measure absorbance of the formazon product was 450 nm, and the reference wavelength was 650 nm. PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31067