In order to confirm our hypothesis, we assessed the methylation status of the Axin gene and investigated transcriptional selleckchem Tofacitinib expression of Axin. In addition, we studied the effects of X ray irradiation on expression of Axin, DNMTs, and MeCP2, its effect on the methylation status of the Axin gene, and the associated changes in cell proliferation, invasiveness, apoptosis and tumor progression. Methods Cell culture and X ray treatment Three cell lines of Non small cell lung cancer, including LTEP a 2, NCI H157 and NCI H460 and one cell line of small cell lung carcinoma NCI H446 were cultured in plastic flasks with RPMI 1640 medium containing 10% fetal calf serum at 37 C in a humidified atmosphere. The plastic flasks with lung cancer cells were treated with X ray irradiation using a linear accelerator with a dose of 1Gy and 2 Gy, respectively, according to the previous study.

X ray irradiation was delivered soon after the cell density reached 70 80%. Untreated lung cancer cells were used as a control. After irradiation, the cells were harvested at the appropriate time points and reserved in a refriger ator before being processed for further analysis. Inhibitors,Modulators,Libraries As previously demonstrated, lung cancer cell lines with different histological types usually show different Inhibitors,Modulators,Libraries radio sensitivity. In order to exclude an influence from histo logical type, two adenocarcinoma cell lines with different methylation statuses and expression levels were used in in vitro and in vivo experiments to study the effect of X ray irradiation.

Nested MSP, Real time RT PCR and western blot analysis The genomic DNA from lung cancer cells treated with or without X ray irradiation were isolated by using a DNA extraction Inhibitors,Modulators,Libraries kit according to the manufacturers instructions. Aliquots of DNA samples were treated with a DNA methylation kit. Hyper methylated Axin gene was defined when a distinctive amplicon was demonstrated on gel electrophoresis after methylation specific PCR, while unmethylated Axin gene was designated when no distinctive amplicon was seen after methylation specific PCR and clear Inhibitors,Modulators,Libraries amplicon was produced by unmethylation specific PCR. The primers for PCR reactions are listed in Table 1. Total RNA was isolated from lung cancer tissues and cultured cells with TRIzol Reagent. Real time RT PCR was performed to evaluate the transcripts of Axin. The experiments were performed according to the manufac turers instructions.

Each assay was repeated three times. The PCR primers are listed in Table 1. Mouse monoclonal antibody against DNMT1, B actin, B catenin, and acetylated histone H3 and Inhibitors,Modulators,Libraries rabbit polyclonal antibody against acetylated histone H4, DNMT3B, Axin, MeCP2, Cyclin D1 and MMP 7 were used in Western blot ana lysis. The protein bands on the membrane were visualized using http://www.selleckchem.com/products/Imatinib(STI571).html ECL and quantified using the DNR Bio Imaging System.