Mda mb 231 cell line

MDA-MB-231 cell line was purchased from ATCC and SUM-159 cell line was a gift from Dr. Peiqing Sun (WFU, USA). Boh cells were cultured in high glucose Dulbecco’s Modified Eagle Medium(DMEN)(Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin, and streptomycin. Transfection was performed by using lipo2000 solution and Opti-MEM culture medium according to the manufacturer’s protocols.

Human breast cancers SK-BR-3 (high HER-expressing cells) and MDA-MB-231 (basal HER-expressing cells) cell lines were purchased from ATCC. The ZR-75-1 cell line (which overexpresses HER2 [27 (link)] without gene amplification [28 (link)]) was a generous gift from Dr Elisa Caiola, Mario Negri Institute, Milan. SK-BR-3 cell line (ATCC HTB-30) was cultured in McCoy’s 5a medium (Euroclone) supplemented with 10% Fetal Bovine Serum (FBS). ZR-75-1 cells (ATCC CRL-1500) were cultivated in RPMI-1640 medium (Euroclone) supplemented with 10% FBS. MDA-MB-231 cell line (ATCC HTB-26) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Euroclone) supplemented with 10% FBS.
Cells were cultured in 100 mm dishes at 37 °C in a humidified atmosphere containing 5% CO₂, and when they reached confluence, cells were passaged using a Trypsin-EDTA solution.

MDA-MB-231 cell line was purchased from ATCC (Cat#HTB-26). MDA-MB-231 cell morphological features, proliferation, differentiation, death and migration were examined by Cell-IQ (Chip-man, Tampere, Finland). MDA-MB-231 cells were cultured in RPMI-1640 Media supplemented with 10% fetal bovine serum. About 5 × 10⁴ cells per well were plated on 24-well plates and incubated for 24 hrs. After then, cells were treated with either DETA NONOate at 1, 5, 15, and 30 μM with or without the addition of 0.5 or 2.5 μM cisplatin respectively. Each group had a blank control (1x PBS). The plates were transferred to 37°C incubator for further culture. After 72 hrs, cells were collected for protein analysis and MTT Assay. MTT assay was carried out with MTT Cell Proliferation Assay Kit based on the manufacture protocol (Life Technologies). All assays were performed in triplicate and repeated thrice.

Human colorectal carcinoma cell line HCT116 p53^+/+ and breast adenocarcinoma MDA-MB-231 cell line was from ATCC. HCT116 p53^−/− were kindly provided by Prof. B. Vogelstein (Johns Hopkins University School of Medicine) [27] (link).
Cells culture medium DMEM was from PAN Biotech (Aidenbach, Germany) and fetal bovine serum (FBS) was from Hyclone (Cramlington, UK). The proteasome inhibitor bortezomib was obtained from Santa Cruz Biotechnology (Santa-Cruz, USA). General caspase inhibitor Z-VAD-FMK and inhibitor to lysosomal cathepsins E-64 were from MP Biomedicals (Eschwege, Germany), Bafilomycin A1 was from Cayman Chemicals (Ann Arbor, USA). Neutralizing antibodies to TRAIL death and decoy receptors were from Enzo Life Sciences (Farmingdale, USA) and R&D systems (Minneapolis, USA), respectively. FITC-conjugated antibodies to DR4, DR5, DcR1 and DcR2 receptor were obtained from Abnova (Walnut, USA), isotype control antibody was from Immunotech (Marcelle, France). For western blot biotinylated goat antibodies to TRAIL death and decoy receptors were purchased from R&D Systems (Minneapolis, USA), HRP streptavidin and antibodies to actin were from Sigma-Aldrich (St. Lotus, USA).

The human TNBC MDA-MB-231 cell line (ATCC HTB-26) and the non-cancerous breast cell line MCF-12A (ATCC CRL-10782) were purchased from ATCC (Manassas, VA, USA). DMEM-HG cell growth medium supplemented with 10% (v/v) FBS was used to culture breast cancer cells. To establish a cisplatin-resistant cell strain, MDA-MB-231 cells were continuously treated with increasing concentrations of cisplatin (up to 2 µM) during a period of 6 months. When a consistent cell growth rate in the presence of cisplatin was achieved, these cells were named MDA-MB-231/R and stocked to assure the consistency of the phenotype for future use and experiments. All posterior experiments were performed within 10 passages to maintain the resistance while routinely growing the MDA-MB-231/R cell line in a cell culture medium without the addition of cisplatin. MCF-12A cells were cultured in DMEM/F12 medium supplemented with 100 ng/mL cholera toxin, 0.01 mg/mL bovine insulin, 20 ng/mL hEGF, 500 ng/mL hydrocortisone and 5% (v/v) horse serum. Cells were grown in monolayers in a sterile environment at 37 °C with a 5% CO₂ humidified atmosphere. Under these conditions, the population doubling time was 25.5 ± 0.9 h and 30.6 ± 1.1 h for MDA-MB-231 and MDA-MB-231/R cells, respectively, and 20.6 ± 3.1 h for MCF-12A cells. The cell cultures were routinely screened for mycoplasma contamination, yielding negative results.

MDA-MB-231 cell line (ATCC HTB-26, Manassas, VA, USA), a human breast adenocarcinoma cell line representative for TNBC, was maintained in culture at 37 °C in humidified atmosphere with 5% CO₂. The cells were cultured in 75 cm² culture flasks using Dulbecco’s Modified Eagle Medium (DMEM, cat. no. 31600-083, Gibco by Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 3.5 g/L glucose, 1.5 g/L NaHCO₃, 1% antibiotics and antimycotics solution (cat. no. A5955, Sigma-Aldrich, St. Louis, MO, USA) and 10% foetal bovine serum (cat. no. 10270-106, origin South America, Gibco by Thermo Fisher Scientific, Carlsbad, CA, USA). Similar, normal MRC-5 cells (human lung normal fibroblasts) (ATCC CCL-171, Manassas, VA, USA) were cultivated in Eagle’s Minimum Essential Medium (MEM, cat no. 41500-018, Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 1.5 g/L NaHCO₃, sodium pyruvate 100 mM (cat. no. 11360-039, Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA), 1% antibiotics and antimycotics solution (cat. no. A5955, Sigma-Aldrich, St. Louis, MO, USA) and 10% foetal bovine serum (cat. no. 10270-106, origin South America, Gibco by Thermo Fisher Scientific, Carlsbad, CA, USA).