Zr 75 1

The ZR-75-1 (ATCC #CRL-1500 pg, RRID: CVCL_0588), T-47D (HTB-133, RRID: CVCL_0553), and MDA-MB-453 (HTB-131, RRID: CVCL_0418) breast cancer cell lines were obtained from American Type Cell Culture Collection (ATCC; Manassas, VA, USA), and the MFM-223 cell line was obtained from the DMSZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany, RRID: CVCL_1408). All cell lines were routinely tested for mycoplasma infection and authenticity confirmed by short tandem repeat profiling (Cell Bank Australia). ZR-75-1 and T-47D cells were maintained in RPMI-1640 medium (Invitrogen) containing 10% Fetal Bovine Serum (FBS) and 2 nM L-Glutamine (Sigma). MFM-223 cells were cultured in EMEM (Sigma) containing 10% FBS, 2 nM L-Glutamine (Sigma), 1 × Non-essential Amino Acids (Sigma), and 1 × Insulin–Transferrin–Sodium Selenite (Sigma). MDA-MB-453 cells were maintained in DMEM (Sigma) medium containing 10% FBS, 2 nM L-Glutamine (Sigma) and 1 × Sodium Pyruvate (Sigma). All lines were incubated at 37 °C and 5% CO₂.

The human breast cancer cell lines MDA-MB-231 (HTB-26), MCF7 (HTB-22), and ZR-75-1 (CRL-1500) were purchased from ATCC (Manassas, VA, USA) and cultured in a humidified cell culture chamber in RPMI (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific) and Anti-Anti (Thermo Fisher Scientific), at 37 °C under a 5% CO₂ atmosphere. Cultures were routinely checked for mycoplasma contamination using the EZ-PCR Mycoplasma Test kit (Biological Industries, Cromwell, CT, USA). Cultures were not used beyond 6 months after thawing. Al experiments were performed within 2 years after purchase of cell lines. For ASO transfection experiments, cells were seeded at 50,000 cells/well in a 12-well-plate (Nunc, Thermo Fisher Scientific). On the next day, cells were transfected with 200 nM (or 300 nM for ZR-75-1) ASO control (ASO-C: 5’-AGGTGGAGTGGATTGGGG) or Andes-1537 (5’-CACCCACCCAAGAACAGG) and 2 μg/ml Lipofectamine2000 (Invitrogen) or left untreated for 24 h. All ASOs contained 100% phosphorothioate internucleosidic bonds (Integrated DNA Technologies, Coralville, IA, USA). For mimic transfection, cells were seeded as above and transfected the next day with 0.5 nM control or specific mimic (Exiqon, Qiagen, Hilden, Germany) and 1 μg/ml Lipofectamine2000 (Thermo Fisher Scientific) and left for 48 h before processing.