Incubated the inserts at 37°C for 4 h for gelling and then pretreated with serum-free medium at 37°C for 1 h before seeding cells at a density of 2 × 104 /ml with 1% FCS. The lower chambers of the transwells were filled with 600 ul medium containing ZD1839 manufacturer 10% FCS. Then the transwell were incubated at 37°C with 5% CO2 for 24 h to allow cells to migrate. After that, removed the cells on the upper side by wiping with cotton

swab. Cells that had invaded through matrigel were fixed in paraformaldehyde and crystal violet stained according to the manufacture’s instruction. Cells that had invaded the matrigel and reached the lower surface of the filter were counted under a light microscope at a magnification of 200×. We chose five fields of vision and counted the MK0683 numbers of the invaded cells and the results from three separate chambers were then averaged. The experiment was

performed in triplicate. Statistical analysis The cell culture data from at least three independent experiments were expressed as means ± SD and examined by one-way analysis of variance followed by the Student–Newman–Keuls test. A Pearson’s correlation test was performed to examine the MX69 ic50 relationship of LRIG1 and EGFR expression in bladder cancer and non-neoplastic tissues. All P-values were two-sided, and values less than 0.05 were considered significant. SPSS v16.0 software was used for all statistical procedures. Results Expression of LRIG1 and EGFR mRNA and protein in bladder cancer and normal tissue In order to examine the mRNA expression of LRIG1 and EGFR in bladder cancer, 45 tumor RNA samples and corresponding 5 normal tissues RNA samples were analyzed by quantitative real-time RT-PCR. Compared with corresponding nonneoplastic tissue, the expression

of LRIG1 appeared downregulated in all of the tumor (Figure 1A). Meanwhile, the expression of EGFR was elevated in all of the tumor compared to the mean in the respective non-neoplastic tissue (Figure 1A). Next, expression of LRIG1 and EGFR protein were determined by IHC. IHC staining also demonstrated downregulation of LRIG1 see more protein in bladder cancer tissue (Figure 1B). Then we compared the expression of LRIG1 and EGFR in different stage. We found that the LRIG1 expression in T2-T3 stage were significantly lower than that in T1 stage. This phenomenon could indicate that the expression of LRIG1 were lower in aggressive bladder cancer. Figure 1 Expression of LRIG1 and EGFR mRNA and protein in bladder cancer and normal bladder tissue. A: LRIG1 and EGFR mRNA expression in bladder cancer with different tumor (T) stages and normal bladder tissue. *P < 0.05 vs normal tissue. #P < 0.05 vs T1 stage. B: Immunohistochemical analysis of LRIG1 and EGFR expression in bladder cancer with different tumor (T) stages and normal bladder tissue.