Mda mb 231

Human breast cancer cell lines MCF7 and MDA-MB-231, and human colon carcinoma cell line HCT116 were obtained from the American Type Culture Collection (ATCC, Manassan, VA, USA). The cells were cultured in Dubecco’s modified Eagle medium (DMEM, HyClone), supplemented with 10% fetal bovine serum (FBS, HyClone), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37 °C with 5% CO₂. UEV1A-overexpressed stable MDA-MB-231 and MCF7 cell lines were created as previously reported [6 (link)]. Stable Uev1A-knockdown cell lines were created by transfecting MDA-MB-231 and MCF7 cells with Uev1A shRNA lentiviral particles or negative control shRNA lentiviral particles-A (Santa Cruz Biotechnology, Inc), and selecting with 1 μg/mL puromycin dihydrochloride (Santa Cruz Biotechnology, Inc).

All cell lines were purchased from ATCC (Rockville, MD, USA) and kindly provided by Dr S. Füssel, Urology Laboratory, University of Dresden (RT-4, UM-UC-3, HT1197, J82, T24, SW1710, and VM-CUB1 bladder cancer cell lines) and by the Department of Obstetrics and Gynecology, University of Rostock (MCF-7, MDA-MB231, BT549, and T47D breast cancer lines). Cells were maintained at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (high glucose, 4.5 g/l) containing 2 mM l-glutamine, 1 mM sodium pyruvate, supplemented with 10% FCS, 0.1 mM non-essential amino acids, 50 U/ml Penicillin and 50 µg/ml Streptomycin. Breast cancer BT549 cells were grown in RPMI medium with the same supplements. RT-4 and MCF-7 cells were incubated with growth factors (EGF, FGF2, TGFB1, from R&D Systems, at 10 ng/ml) for 48 h prior to experiments. UM-UC-3 and MDA-MB231 cells were treated with inhibitors (EGFR inhibitor Tyrphostin AG 1478, FGFR1 inhibitor PD161570, TGFBR inhibitor SB431542, from Santa Cruz, at concentrations ranging from 200 nM to 3 µM) for 24 h prior to Boyden chamber assays. We used non-invasive RT-4 bladder and MCF-7 breast and invasive UM-UC-3 bladder and MDA-MB231 breast cancer cell lines to model the EMT transitions. All cell lines were tested for mycoplasma contamination prior to the experiments according to manufacturer’s instructions (Venor GeM Classic, Minerva Biolabs).