Umuc3

Human BCa cell lines (T24, UMUC-3, and 5637) and normal human urothelial cell line (SV-HUC-1) were obtained from the American Type Culture Collection. The human BCa cell line BIU-87 was obtained from the China Center for Type Culture Collection. BIU-87 and 5637 cells were cultured in RPMI-1640 medium (Servicebio) supplemented with 10% fetal bovine serum (FBS) (Biological Industries) and 1% penicillin/streptomycin (Solarbio, China). T24 and UMUC-3 were cultured in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% FBS. SV-HUC-1 cells were cultured in F-12 K medium (iCell-0007) supplemented with 10% FBS (Biological Industries) and 1% penicillin/streptomycin (Solarbio, China). All cell lines were cultured at 37 °C in a 5% CO₂ standard incubator.

Human BC cell lines T24, UM-UC-3, RT4, J82, 5637, HT-1376, TCCSUP and the immortalized normal uroepithelium cell line SV-HUC-1 were purchased from American Type Culture Collection (ATCC, USA). T24 and 5637 cells were cultured in RPMI-1640 (Gibco, Shanghai, China), RT4 cells were cultured in McCoy’s 5A(Gibco), UM-UC-3, J82, HT-1376, TCCSUP and HEK-293 T cells were cultured in DMEM (Gibco), whereas SV-HUC-1 cells were maintained in F-12 K media (Gibco), supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco). Cells were grown in a humidified atmosphere of 5% CO₂ at 37 °C. For Actinomycin D assay, T24 and UM-UC-3 cells were exposed to 2 μg/mL Actinomycin D (Sigma, USA) to block transcription for 8, 16 and 24 h.

The human cell lines 5637, T24, RT4, J82, SW780, UMUC-3, A549, and SK-OV-3 were obtained from the American Type Culture Collection (Maryland, USA). All cell lines were authenticated by STR profiling and tested for mycoplasma contamination. 5637, T24, SW780, and A549 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). RT4, J82, and UMUC3 cells were grown in DMEM/F12 supplemented with 10% FBS. SK-OV-3 cells were grown in McCoy’s 5a medium with 10% FBS. The sorted BCSCs (as described below) were maintained in serum-free DMEM/F-12 supplemented with 20 ng/mL EGF (#PHG0311, Gibco), 20 ng/mL FGF-basic (#13256–029, Gibco), 1% N-2 (#17502001, Gibco), and 2% B-27 (#17504044, Gibco). BCSCs were passaged less than 10 times in vitro. Primary bladder cancer cells (pBCs) were isolated from a chemotherapy-naïve patient. Briefly, fresh bladder tumors were minced into small fragments and digested with 200 U type I collagenase (#07415, STEMCELL Technologies), 0.01% hyaluronidase (#07461, STEMCELL Technologies), and 0.01% DNase I in RPMI 1640 at 37°C for 120 min. Subsequently, cells were filtered through a cell strainer (70 μm), washed, suspended with phosphate-buffered saline (PBS) to yield single cells, or cultured in complete medium for further experiments.

The human bladder cancer cell line, UM-UC-3, was purchased from the American Type Culture Collection (Manassas, VA). Wild-type UM-UC-3 and its sublines were established in orthotopic xenograft models (primary bladder cancer, and lung, liver, and bone metastatic cells) cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco^®-life Technologies, Grand Island, NY, USA). Flufenamic acid and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO). Human recombinant interleukin (IL)-6 and transforming growth factor-β1 (TGF-β1) were obtained from R&D Systems (Minneapolis, MN), and human recombinant IL-1β was purchased from PeproTech (Rocky Hill, NJ). Antibodies were purchased from the following suppliers: the antibody to aldo-keto reductase 1C1 (AKR1C1) was from Lifespan Biosciences (Seattle, WA); those for Snail, Slug, N-cadherin, phospho-FAK (Y397), phospho-Akt (S473), and phospho-Src (Y416) were from Cell Signaling Technology (Beverly, MA); those for fibronectin and Rac1 were from BD Transduction; and that for α-tubulin was purchased from Sigma-Aldrich.

TCC cell lines T24 and UMUC-3 were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM). Culture medium was supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 0.1 mg/ml streptomycin (Gibico). Cells were incubated in a humidified atmosphere contains 5% CO₂ at 37°C and observed by inverted microscope (×100 and ×200, Olympus).

Human BC cell lines (UM‐UC‐3 and 5637) were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco BRL), supplemented with 10% foetal bovine serum (Gibco BRL) and penicillin/streptomycin at a concentration of 100 U/mL. The cultured cells were maintained at 37°C in a humidified atmosphere with 5% (v/v) CO₂.
Melittin was purchased from Sigma–Aldrich Corp. BC cells were plated in 24‐well plates (5 × 10⁴ cells/well). Subconfluent cells were treated with MEL at various concentrations (0, 2, 4 and 6 μg/mL) for 24 hours.