Three independent experiments were carried out for each treatment. Flow cytometric analysis Eca109

and Y-27632 manufacturer Kyse510 (4 × 105) were seed in 12-well plates and then were transfected. Transfected Cells were Selleckchem Cl-amidine harvested at 24 h, 48 h, and 72 h for flow cytometric analysis. Cells were washed twice with PBS and then incubated with 20 ug/ml PI, 100 ug/ml RNase, and 0.1% triton X-100 in PBS for 30 min in the dark. The PI stained cells were analyzed for cell cycle distribution and apoptosis by using a FACScalibur instrument (BD bioscience, San Jose, A) equipped with Cell Quest software (Becton Dickinson). Statistical analysis Students’s t-test for equality of means was used to compare values. Person’s correlation coefficient was used to determine the relationship. P values less than 0.05 were considered significant. All analyses were performed with SPSS version 16.0 software.

Results Overexpression of GADD45α in tumor tissue of ESCC The mRNA expression levels of GADD45α, GADD45β, GADD45γ in tumor tissue and adjacent normal tissue from ESCC were detected. GADD45α mRNA level was higher in tumor tissue than in adjacent normal tissue (P = 0.001) (Figure 1A and Table 3). No significant difference was found in GADD45β(Figure 1B and Table 3) and GADD45γ(Figure 1C and Table 3)mRNA levels between tumor and adjacent normal tissue. The overexpression of GADD45α in tumor tissue of ESCC was confirmed at the protein level using immunohistochemistry (Figure 1E,F and 1G) and western blotting (Figure 1H). GADD45α-positive PtdIns(3,4)P2 staining was mainly located in nucleolus AZD0156 nmr of tumor

cells with few positive staining in surrounding matrix. To show the statistical discrimination clearly, samples with nuclear GADD45α-IRS < 5 were classified as GADD45α -negative (Figure 1F), and those with GADD45α-IRS > 5 were classified as GADD45α positive (Figure 1E), the ratio of GADD45α positive was higher in tumor tissues than normal tissues (Table 4). Figure 1 Growth arrest and DNA damage-induced 45a (GADD45α), GADD45β, GADD45γ gene expression in tumor tissue compared with adjacent normal tissue from the same esophageal squamous cancer patients. A, B and C, Relative expression of GADD45a, GADD45β, GADD45γ mRNA in tumor tissues from ESCC patients was measured by quantitative real-time PCR. Results were normalized to the level of β-actin (loading control). D shows the different expression levels of GADD45α in various TNM stages. G. Protein levels of GADD45α in tumor tissue and adjacent normal tissue from ESCC patients were assessed by immunohistochemistry. E shows the representative GADD45α-positive staining in tumor tissue from ESCC patients. GADD45α protein is mainly located in nucleolus of tumor cells. F. Negative control with less GADD45α staining in normal tissue. H Protein levels of GADD45α in tumor tissue and adjacent normal tissue from ESCC patients were assessed by western blotting.