5 h at room temperature with peroxidase-linked secondary antibody (Roche), and signals were detected using Lumilight Plus Western blotting kit reagents (Roche) according to the manufacturer’s instructions and luminescence imaging (LAS-1000, Fujifilm). Statistical analysis We used the χ2 and Fisher’s exact tests to evaluate the differences of staining of E-cadherin and Snail, Slug and Twist according to patient and RGFP966 cancer characteristics. The overall survival was

defined as the time between the date of surgery and the last date of follow-up or date to death owing to bladder cancer. The progression-free survival was defined as the time interval between the date of surgery and the date of progression/recurrence or date of last follow-up. The curves were done using the Kaplan-Meier method with the log-rank test to assess the selleck products statistical significance. Cox proportional hazards analysis was used to determine LGK-974 cell line the relative contribution of various factors to the risk of death,

recurrence, and progression. P < 0.05 was considered as statistically significant. Analyses were performed with SPSS 10.00 software (SPSS, Chicago, IL). Results Expression of Snail, Slug, Twist and E-cadherin in human bladder cancer cell lines The expression of Snail, Slug, Twist and E-cadherin was analyzed at the mRNA and protein level by semiquantitative RT-PCR(Fig. 1A) and western blot (Fig. 1B) in the human bladder cancer cell lines T24, HTB-3, HTB-1, HTB-2 and HTB-9. Slug was expressed with different intensities in all five cancer cell lines. The undifferentiated HTB-1 and T24 cells had a strong mRNA and protein expression of Slug, whereas the other 3 cell lines showed only weak expression levels. Twist mRNA and protein was detected in HTB-1 and T24 cells, no appearant Twist mRNA and protein

expression was found in other 3 cell lines. E-cadherin was detected in Adenosine HTB-2, HTB-9 and HTB-3 cell lines. The most undifferentiated cell line HTB-1 and T24 cells showed no E-cadherin expression. Snail was not detectable in all five cancer cell lines. To verify intact RNA and protein, β-actin was used as a positive control. Figure 1 Expression of Snail, Slug and Twist in five bladder cancer cell lines T24, HTB-1, HTB-2, HTB-3 and HTB-9. The analysis of the relative mRNA and protein intensity of Slug, Snail and Twist compared with E-cadherin showed that bladder cancer cells with a high Slug and Twist expression had no or only low E-cadherin expression. In contrast, cells with low Slug and Twist expression had high expression levels of E-cadherin. Expression of Snail, Slug, and Twist in correlation with E-cadherin in human bladder cancer tissue Slug(A), Twist(B, F), Snail (Fig. 2C and 2G) in primary bladder cancer tissue were identified in the cytoplasm as well as in the nucleus of cancer cells. In general, staining for Slug and Twist was more intense than for Snail.