Finally, the me dium was aspirated very carefully and 150 ul/well of DMSO was added to dissolve for mazan crystals. The absorbance of each well was obtained using a plate reader at a test wavelength of 490 nm with a reference wavelength of 630 nm. The value of treatment group was always normalized to that of control group. Scratch assay As described, selleck inhibitor twelve well plates were pre coated with poly lysine, followed by further BSA blocking. A sufficient number of PaTu8988 cells were plated, so that they became confluent in the wells right after attachment. Same area of each well is then displaced by scratching a same straight line through the layer with a needle. Floating cells were washed away by warm PBS. Cells were further incubated with the indi cated concentration of SAHA for 24 h, and stained with Wright Giemsa to see migration gap.

Mitomycin C was always included in the culture media to prevent cell proliferation. PCR analysis Total RNA was extracted from PaTu8988 cells and trea ted with RNase free DNase I. The quality of RNA was test by DU 800 Nucleic Acid/Protein Analyzer. The cDNA was generated by reverse transcrip Inhibitors,Modulators,Libraries tion using RevertAidTM First Strand cDNA Synthesis Kit and oligo in a 20 uL reaction Inhibitors,Modulators,Libraries containing 5 ug of total RNA. Next, PCR was performed in each 25 uL PCR reaction containing 0. 5 uL diluted cDNA, TaKaRa rTaq DNA Polymerase and indicated primers. The PCR reaction contained an initial denaturation at 94 C for 3 min, followed each PCR cycle by de naturation at 94 C for 30 seconds, annealing at 55 68 C for 30 sec onds, and extension at 72 C for 1 min for a total of 22 36 cycles, depending on the primer Inhibitors,Modulators,Libraries length and the molecular weights of target genes.

PCR products were an alyzed Inhibitors,Modulators,Libraries by 1. 5% agarose gel. Primers used in this study were summarized Inhibitors,Modulators,Libraries in Table 1. Western blot analysis As described before, aliquots of 30 40 ug of protein from each sample was separated by 10% SDS polyacrylamide gel electro phoresis and transferred onto a polyvinyli dene difluoride membrane. After blocking with 10% instant nonfat dry milk for 1 h, membranes were incubated with the specific antibody overnight at 4 C, followed by incubation with corresponding secondary antibody for 30 min to 1 h at room temperature. Antibody binding was detected with the enhanced chemiluminescence de tection system.

The intensity of interested band was quantified sellckchem using Ima geJ software, and the value was normalized to correspond ing loading controls. Statistic analysis The data shown in this study represented the mean S. E. Differences between the groups were assessed by one way ANOVA using SPSS 16. 0 software. The significance of dif ferences was indicated as P 0. 05 and P 0. 01. Results SAHA inhibits the proliferation of PaTu8988 pancreatic cancer cells Figure 1A showed the chemical structure of SAHA.