SW480 or HCT 116 cells were plated in 96 well plates and incubated for 24, 48, 72, 96 h respect ively after transfection. 20 ul of 5 http://www.selleckchem.com/products/Gemcitabine-Hydrochloride(Gemzar).html mg mL MTT was added into each corresponding test well, and incubated for 4 h in 37 C incubator. The supernatant was then discarded, and 200 ul of DMSO was added to each well to dissolve the formazan. Optical density was evaluated by measuring the absorbance. Inhibitors,Modulators,Libraries The absorbance at 570 nm of each well was read on a spectrophotometer. All experiments were performed in triplicate. Apoptosis assay The apoptosis ratio was analyzed using the Annexin V FITC Inhibitors,Modulators,Libraries Apoptosis Detection Kit. At 72 h after transfection cells were harvested and resuspended in binding buffer containing Annexin V FITC and PI according to the manufacturers instruc tions. The samples were analyzed by flow cytometry.

Inhibitors,Modulators,Libraries Cells were discrimi nated into viable cells, necrotic cells, and apoptotic cells by using BD FACSDiva 6. 1. 3 software, and then the percentages of apoptotic cells from each group were compared. Tests were re peated in triplicate. Wound healing assay SW480 cells or HCT 116 cells were seeded onto 6 well plates. When the cell confluence reached about 80% and above at around 48 h post transfection, scratch wounds were made by scraping the cell layer across each culture plate using the tip of 10 ul pipette. After wounding, the debris was removed by washing the cells with PBS. Wounded cultures were incubated in serum free medium for 36 h, and then 3 fields were randomly picked from each scratch wound and visualized by mi croscopy to assess cell migration ability.

The experi ments were performed in triplicate. In vitro transwell invasion assay Transwell membranes coated with Matrigel were used to assay cell invasion in vitro. Inhibitors,Modulators,Libraries At 48 h post transfection, cells were resuspended into serum free medium. Transfected cells were reseeded into the upper chamber, and 0. 6 ml medium with 10% FBS was added to the lower chamber as chemoattractant. After 24 h incubation, non invading cells on the upper surface of the membrane were removed with a cotton swab. The invasive cells, which penetrated to the lower surface, were fixed with 4% paraformaldehyde and stained with 0. 1% crystal violet. The number of cells invading the membrane was counted from 5 ran domly selected visual fields with an inverted microscope at 100 magnification. Data were obtained from 3 inde pendent experiments.

Inhibitors,Modulators,Libraries Statistical analysis Experimental data were presented as the mean standard deviation. All statistical analyses were performed using T test when only 2 groups were compared, and by ANOVA when 3 or more groups were selleck compound compared. All ana lyses were performed with SPSS 19. 0, and a value of P 0. 05 was considered to indicate statis tical significance. Background Pancreatic cancer is the fourth leading cause of cancer death, and is amongst the deadliest of human cancers.