Tumor recurrence/progression was defined based on clinical, radiological, or histological diagnoses. The study was approved by the affiliated hospital of Qingdao medical college Faculty of Medicine Human Investigation Committee. Table 1 Clinical information of patient samples analyzed    Variable n (%) Tissue type      Background 42    Tumor 120 Age – yr (mean)   < 70 64 (53) ≥70 56 (47) Gender GDC-0973 cost – number of patients

  Male 87 (72) Female 33 (28) Grade – no. of patients   LG 41 (34) HG 79 (66) Stage – number of patients (%)   NMIBC:   Ta 31 (26) T1 45 (37) MIBC:   T2N0M0 23 (19) T3 N0M0 19 (16) T4/Any T N+/M+ 2 (1.6) Surgical procedure   TUR 76(63) Cystectomy 44(37) Recurrence   Number of patients with NMIBC 23(19) Progression: Number of patients with NMIBC 8 (6.6) Number of patients with MIBC 15 (12.5) Survival Number of patients with MIBC      Cancer-specific   Alive 27 (22.5) Deceased 17(14)    Overall survival   Alive 25 (21) Deceased 19 (16) Immunohistochemistry Immunohistochemical

staining was done on paraffin-embedded tissue, which had described in detail before[16]. Briefly, three-micrometer-thick sections were cut, using a rotation microtom. The sections were deparaffinized in xylene and rehydrated in graded alcohols and distilled water. After antigen retrieval with 0.01% EDTA (pH 8.0), endogenous peroxidase activity was blocked PS-341 manufacturer with 1% hydrogen peroxide in distilled water for 25 min followed by washing with distilled water and finally

PBS + 0.1% Tween for 5 min. To bind nonspecific antigens, the sections were incubated with 1× Power Block (BioGenex) for 5 min. The primary antibodies for Snail, Slug, Twist, and E-cadherin were either polyclonal rabbit anti-Twist and anti-E-cadherin or polyclonal goat anti-Snail and anti-Slug, Baf-A1 and purchased from Santa Cruz Biotechnology. Antibody dilution ranged from 1:50 to 1:150 in PBS for 30 min at 37°C. As negative control, sections were incubated with PBS instead of the primary antibody. This was followed by incubation with biotinylated antirabbit/antigoat immunoglobulin G (1:200; Santa Cruz Biotechnology) for 30 min at 37°C and peroxidase-conjugated avidin-biotin complexes (KPL) and 3,3′-diaminobenzidine (Sigma). The sections were then counterstained with Mayer’s hematoxylin, upgraded alcohols, mounted, and analyzed by standard light microscopy. Evaluation of immunohistochemistry results Immunohistochemical staining of Snail, Slug and Twist and E-cadherin was defined as detectable immunoreaction in perinuclear and/or cytoplasm. Expression of Snail, Slug and Twist was considered negative when no or less than 49% of the tumour cells were stained[16]. Cancer cells that were immunostained less than 10% staining were defined as having a reduced E-cadherin expression[17]. Cell lines The human bladder cancer cell lines (T24, HTB-3, HTB-1, HTB-2 and HTB-9) obtained from ATCC (Rockville, MD, USA).