The primers for detection of the methylated and unmethylated DACT2 CpG islands were described in the previous study. For bisulfite ge nomic sequencing, 2 ul of bisulfite treated DNA was amplified using primers. The PCR products were example cloned into a PUCm T vector. Three Inhibitors,Modulators,Libraries to five colonies were randomly chosen and sequenced for plasmid DNA extraction with QIAprep Spin Miniprep Kit. RNA interference Short interfering RNA duplexes were used to downregulate DACT2 ex pression, and a nonsilencing siRNA was used as negative control. After growth in the plate for 24 h, cells were transfected with 33 nM siRNA using Lipofectamine 2000 transfection reagent according to the manufacturers instructions. The efficacy of transfec tion was checked by real time PCR after 48 h.

Cell cycle analysis MHCC97L cells were transfected with DACT2 siRNA or a negative control. After 48 h, cells were harvested and stained with a DNA PREP kit. The percentage of cells in G0G1, S, and G2M phase was quantified using flow cytometry analysis according to the manufacturers instructions. Analysis of cell cycle data was performed with Multicycle analysis software. All experiments Inhibitors,Modulators,Libraries were completed in triplicate. Cell invasion and motility assay Cell invasion analysis was performed using 24 well transwell plates. Forty eight hours after RNA interference, the filters were coated with Matrigel in the upper Inhibitors,Modulators,Libraries compartment and seeded with 200 ul of 0. 8 105 cells. The lower compartment was filled with cell culture medium supplemented with 15% fetal bovine serum. After 48 h, migrated cells on the bottom surface were fixed with methanol and stained with 0.

1% Crystal Vio let. The cell motility Inhibitors,Modulators,Libraries assay was performed similarly, ex cept the cells were applied into the uncoated filter and incubated for 24 h. The invading cells were examined, counted, and photographed using digital microscopy. Three fields were counted per filter in each group. Statistical analysis The relationship between DACT2 expression and clinico pathological variables were assessed by the chi square test. The detailed statistical tests used in the study are shown in Results and in the table and figure legends. P 0. 05 was considered statistically significant. Results Analysis of DACT2 expression in clinical samples Expression of DACT2 mRNA in HCC tumor specimens was lower than in nontumoral liver specimens in 24 of the 30 paired cases.

The average level of DACT2 mRNA expression in HCC tissues was 2. 25 fold lower than in adjacent noncancerous tissues as shown in Figure 1A. DACT2 protein Inhibitors,Modulators,Libraries sellectchem expression was also investigated by immunohistochemis try in tumor tissues and the nontumoral liver counterparts, as is shown in Figure 1B,C,D. In healthy liver tis sues, DACT2 was almost homogeneously expressed in non neoplastic hepatocytes. In tumor tissues, two staining patterns could be distinguished. In most patients, DACT2 expression was markedly decreased in tumor tissues compared with nontumorous liver tissues.