C with mixture of the following primary anti bodies, anti pRelA and anti STAT3. Alexa fluor 555 conjugated anti rabbit IgG and ?488 conjugated anti mouse IgG were used as secondary antibodies. Cells were stained with 40,6 diamidino selleck catalog 2 phenylindole, which was used for nuclear staining. Immuno fluorescence was detected with a fluorescence microscope. Wound healing assay Cells were cultured in 6 well plates until confluent. The cell monolayer was scratched with a sterile pipette tip to generate a wound. The remaining cells were washed twice with culture medium to remove cell debris. Spon taneous cellular migration was monitored using a phase contrast microscope and captured using an Olympus Digital Camera at 0, 24 and 48 h. The area of the scratches was measured and quan tified using NIH Image Analysis software.
A 24 well Insert System using an 8 um polyethylene ter ephthalate membrane was obtained and coated with Matrigel. Inserts were rehy drated with RPMI1640 for 2 h at room temperature prior to use. After rehydration, media was removed and cells were added to the top of each insert chamber in RPMI1640 containing 1% FBS. Lower cham ber contained the medium with 10% FBS as a chemo attractant. After incubation for 48 h, non invading cells were carefully removed from the top of each insert with a cotton swab. Invasive cells were stained with 0. 2% crystal violet in 20% methanol as described previously and were observed with an inverted microscope. Stained cells also dissolved in 10% SDS, and absorbance was measured at 570 nm using an ELISA reader.
Statistical analysis For tissue array analysis, statistical analyses were con ducted using SPSS version 11. 0 statistical software pro gram, and the chi squared test was used to determine the correlations between the expressions of NF ��B, pSTAT3, and MMP9. For cell cul ture experiments, data were analyzed using GraphPad Prism software for Windows Vista and the two tailed Stu dents t test was used to determine the significances of the results. P values of 0. 05 were considered statisti cally significant for all statistical analyses. Results NF ��B, pSTAT3 and MMP9 are positively correlated with each other in clinical gastric cancer specimens Representative results of the immunohistochemical stain ing are shown in Figure 1. Immunoreactivity for NF ��B and pSTAT3 were found in both the nuclei and cytoplasm of tumor cells.
Cells showing distinct nuclear staining, regardless of the presence of cytoplasmic staining, were considered to express Drug_discovery activated forms of NF ��B or STAT3. On the other hand, the expression of MMP9 was detected Cisplatin mainly in the cytoplasm of tumor cells. Positive immunoreactivity for nuclear NF ��B was found in 41 of 255 of clinical samples of gastric cancer. In addition, the expression of nuclear pSTAT3 and cytoplasmic MMP9 were found in 61 of 255 and 46 of 255 of gastric cancer speci mens, respectively. Data concerning the correlations between NF ��B activation, STAT3 activation, and MMP9 expression