STAT3 Signaling in Pancreatic Cancer Figure 3. EGCG inhibits the expression of STAT3-regulated genes. (A), STAT3 activity. AsPC-1 and PANC-1 cells were transfected with pGreenfire1-STAT3 reporter plasmid. Cells were treated with EGCG (0–80 mM). After incubation of 24 hours, luciferase activity was determined using the Dual-Luciferase Reporter Assay System, according the manufacturer’s instructions on a multilabel plate reader. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. (B), VEGF, Bcl-XL, c-Myc, Survivin and Cyclin D1 were detected by qRT-PCR. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0031067.g003 Caspase-3/7 Assay Cells (36104 per well) were seeded in a 96-well plate with 200 ml culture medium. Approximately 16 h later, cells were treated with various doses of EGCG. Casapse-3/7 activity was measured as per manufacturer’s instructions (Invitrogen). Scratch assay AsPC-1 scrambled and STAT3 shRNA cells were seeded in 6 well dishes. When all the cultures were 50% confluent, a cross was marked in the center of each dish using a 10 ml tip. The cells were washed with PBS and cultured in fresh medium. The scratch pictures were taken under a fluorescence microscope at 0, 24 and 48 h for the same positions after the cells were treated with EGCG. Transwell migration assay To determine the effect of EGCG on cell migration, AsPC-1 and PANC-1 cells were plated in the top of chamber onto the noncoated membrane (24-well insert; pore size, 8 mm; Corning Costar) at a density of 16104 cells/well in RPMI medium containing 1% FBS, and allowed to migrate toward RPMI medium containing 10% FBS in the lower chamber. EGCG was added to the both chambers to achieve the concentration of 0, 20, 40, 60 mM, respectively. After 24 h of incubation, cells were fixed with 4% paraformaldehyde and stained with crystal violet. The migrated cells were counted under a light microscope (four random fields per well). Transwell invasion assay To determine the effect of EGCG on cell invasion, AsPC-1 and PANC-1 cells were plated in the top of chamber onto the Matrigel coated membrane (24-well insert; pore size, 8 mm; Corning Costar) at a density of 16104 cells/well in RPMI medium containing 1% FBS, and allowed to invade toward RPMI medium containing 10% FBS in the lower chamber. EGCG was added to the both chambers to achieve the concentration of 0, 20, 40, 60 mM, respectively. After 48 h of incubation non-invaded cells were removed by cotton swab, and invaded cells were fixed with 4% paraformaldehyde and stained with crystal violet. The invaded cells were counted under a light microscope (four random fields per well). Transient transfection and STAT3 reporter AsPC-1 and PANC-1 cells were cultured in 100 mm dishes and transfected at 70% confluent with pGreenfire1-STAT3 reporter plasmid using Lipofetamine 2000 (Invitrogen). After 24 h, cells were treated with EGCG (0–80 mM). After incubation of 24 h, luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega), according the manufacturer’s instructions on a multilabel plate reader (Wallac Victor, Perkin-Elmer). RNA isolation and real-time RT-PCR Total RNA was isolated from AsPC-1 and PANC-1 cells using TRIzol reagent (Invitrogen). RNA concentration was determined using Nano Drop 2000 Spectrophotometer. cDNA was synthesized and RT-PCR reactions were performed using SuperScript II (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed on the Applied Biosystems 7300 Real-time PCR System, using the following program: 50uC for 2 min, 95uC for 10 min, and then 40 cycles of 95uC for 15 s and 60uC for 1 min. PCR PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31067