Mcf 7 htb 22 cells

MCF-7 cells (HTB-22) [30 (link)] were purchased from ATCC (Manassas, VA) and maintained in DMEM-F12 complete media supplemented with 10% fetal bovine serum (FBS), MEM nonessential amino acids, gentamicin and 10μg/mL insulin in a 5%CO₂ incubator at 37°C. Media was changed every 2–3 days and cells were passaged when 65–80% confluent. MCF-7 cells were limited to use within the first 10 passages from the original purchased vial from ATCC, to control for genomic drift due to instability. As a comparison, MCF-7 cells purchased from the same lot from ATCC and cultured under the same media conditions were graciously provided by Dr. James Yager of Johns Hopkins University. For 2D cell samples, MCF-7 cells were plated at a density of 300,000 cells/well in 6-well plates and allowed to grow for 3 days. Cells were scraped into Trizol for RNA analysis and stored at -80°C. For imaging studies, all MCF-7 cells were grown on poly-L-lysine coated coverslips for 3 days at a seeding density of 300,000 cells/well in 6 well plates.

The experimental cell lines, mouse 4T1 breast cancer cells (CRL-2539; GFP-expressing cells), human MCF7 breast cancer cells (HTB-22) and human MDA-MB-231 breast cancer cells (HTB-26), were purchased from ATCC. All cell lines were incubated at 37 °C in a 5% CO₂ atmosphere, used for 10 passages after reviving from the frozen vials and were regularly stained with DAPI (Vector Labs) to test for Mycoplasma contamination every 3 months. 4T1 cells were cultured in RPMI 1640 medium (ATCC) with 10% (final concentration) FBS. MDA-MB-231 cells were cultured in RPMI 1640 medium (ATCC) with 20% FBS. MCF7 cells (HTB-22) were cultured in ATCC-formulated Eagle minimum essential medium with 0.01 mg/mL human recombinant insulin and 10% FBS. The cells were preincubated with KR (50 μmol/L). When the cells reached 70–80% confluence, doxorubicin (DOX) (0.425 μmol/L), docetaxel (DOC) (0.9 μmol/L), AMD3100 (30 μmol/L), the neutralizing TGFβ (1, 2, 3) antibody (N-TGFβ) (1.0 μg/mL) and rapamycin (1 μmol/L) were used to treat the breast cancer cells.

MCF-7 cells (HTB-22) were obtained from the American Type Culture Collection (Manassas, Rockville). Cells were grown as monolayer cultures at 37°C and 5% CO₂, in Eagle's minimal essential medium (E'MEM) supplemented with 10% (v/v) FBS (Sigma), 2 mM L-glutamine (Sigma), 20 mM HEPES (Sigma), 0.025 μg/ml amphotericin B (Sigma), 100 IU/ml penicillin G (Sigma), and 100 μg/ml streptomycin (Sigma). Primary fibroblasts were isolated and identified as previously described [34 (link)]. The donor donating tissues gave written informed consent, and this study was approved by the Ethical Committee for Biomedical Research of the Vietnam National University, Ho Chi Minh City (Ref. 1387 QĐ-ĐHQG). Cells were cultured in D'MEM/F12 supplemented with 10% (v/v) FBS, 20 mM HEPES, 0.025 μg/ml amphotericin B, 100 IU/ml penicillin G, and 100 μg/ml streptomycin at 37°C, 5% CO₂.

MDA-MB-231 (HTB-26) cells, obtained from the European Collection of Authentic Cell Cultures (ECACC, Wiltshire, UK), were kindly provided by Prof. Jekaterina Erenpreisa (Latvian Biomedical and Research Centre, Riga, Latvia). MCF-7 cells (HTB-22) were purchased from the American Type Culture Collection (ATCC). Cells were grown under standard conditions (37 °C, 5% CO₂) in DMEM low-glucose (MCF-7; Sigma-Aldrich, St. Louis, MO, USA, D5546) or DMEM high-glucose (MDA-MB-231; Biowest, Nuaillé, France, L0104) medium supplemented with 10% fetal bovine serum (FBS) (Cytogen, Zgierz, Poland, S181H), antibiotic–antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA, A5955) and, in the case of, DMEM low-glucose medium, 2 mM l-Glutamine solution (Sigma-Aldrich, St. Louis, MO, USA, G7513) was added. The cells were seeded 24 h before treatment at a density of 1 × 10⁴ cells/cm². To induce senescence, cells were treated with 100 nM dox (IC30; Sigma-Aldrich, St. Louis, MO, USA, D1515) for 24 h and then cultured in fresh medium without the drug for several days (1 + n). Every third day, the medium was replaced by a fresh one. The escaper cell line was established after a monthly culture of small cells collected on D1+19 for MDA-MB-231 cells and on D1+13 for MCF-7 cells in four independent experiments.

MCF-7 (HTB22) cells were acquired from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). ACHN (CRL-1611) cells were acquired from the ATCC, and maintained in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS. The cells were grown in a humidified incubator that was maintained at 37 °C with 5% CO₂. Demethylating conditions were established by treating the cell lines for 3 days with 0.2 μM Trichostatin-A (TSA) and 5 μM 5-aza-2′-deoxycytidine (5-A-DC) (SIGMA). Daily, the medium was replaced with fresh medium containing 5-A-DC and TSA. The transfection of constructs was performed using Lipofectamine 2000 (Invitrogen). TNF-α and IFN-α were purchased from R&D and PROSPEC, respectively.