Htb 1

Human bladder cancer J82 cells, derived from a poorly differentiated, invasive stage 3 transitional cell carcinoma [25 (link)], were obtained from ATCC (HTB-1; Manassas, VA, USA). J82 cells were maintained in growth medium consisting of MEM supplemented with 10% FBS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2% MEM vitamin solution, and 100 units/mL penicillin-100 μg/mL streptomycin. The UROtsa cell line was isolated from a primary culture of normal human urothelium and immortalized with the SV40 large T antigen [26 (link)]. UROtsa cells were provided by Dr. Ricardo Saban (Department of Physiology, University of Oklahoma Health Sciences Center) and cultured in DMEM/F12 supplemented with 10 ng/mL EGF, 1 × ITS media supplement, and penicillin-streptomycin. Both cell lines were maintained in a humidified cell incubator at 37°C and 5% CO₂ and passaged every 3–4 days or when cells reached about 80% confluence.

Four bladder cancer cell lines, J82 (ATCC^® HTB-1^™), HT-1376 (ATCC^® CRL-1472^™), RT4 (ATCC^® HTB-2^™), and T24 (ATCC^® HTB-4^™) and a normal human urothelial cell line, SV-HUC-1 (ATCC® CRL-9520™), was obtained from ATCC (Manassas, VA, USA). J82 and HT-1376 cell lines were cultured in Eagle's Minimum Essential Medium (EMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma). RT4 and T24 cells were cultured in McCoy's 5a Modified Medium (ATCC, Catalog No. 30-2007) supplemented with 10% FBS. All cells were cultured at 37°C with 5% CO₂.

In order to confirm our results on TUR samples collected from patients, we worked with three immortalized commercially available UC cell lines well known and characterized [34 (link)–36 (link)]: RT4 (ATCC ®HTB-2™), isolated form transitional cell papilloma and classified as differentiated low grade UC; J82 (ATCC HTB-1™), derived from UC high histological grade that infiltrated into deep muscle (Stage T3); and T24 (ATCC HTB-4™), derived from UC high histological grade (Table 1).