Mcf 7

Oestrogen receptor(ER)-positive (MCF-7, ZR-75-1 and T47D), triple-negative (MDA-MB-231, HCC 1806), and ER-negative/HER2-positive (SK-BR-3) human breast carcinoma cell lines and MCF-10A, a non-malignant immortalised mammary cell line, were purchased from European Collection of Cell Cultures (Wiltshire, UK). HMEC, primary human mammary epithelial cells were purchased from Life Technologies (Carlsbad, CA). HEK-293FT cells were purchased from Invitrogen (Carlsbad, CA) as a part of BLOCK-iT Lentiviral RNAi Expression System. HEK293-T cells were obtained from DSMZ (Germany), as reported previously.^{7 (link)} See Supplementary Methods for culturing conditions.

The MCF-7 and MDA-MB-231 human breast adenocarcinoma cell lines and T47D human ductal breast carcinoma cell line were purchased from the European Collection of Cell Cultures (ECACC). The cells were cultured in Dulbecco’s modified Eagle’s medium with (MDA-MB-231) or without (MCF-7 and T47D) phenol red (DMEM, Lonza, Belgium), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Lonza), 4 mM L-glutamine (Lonza), 1% penicillin/streptomycin solution (Lonza) and 10 mM HEPES buffer (Sigma, Germany). The medium without phenol red was used for the two cells lines expressing the estrogen receptor (i.e., MCF-7 and T47D) due to the potential influence of phenol red on estrogen signaling [40] (link). The cells were cultured in 75 cm² tissue culture flasks (TPP, Switzerland) at 37°C in a humidified atmosphere containing 5% CO₂. Sub-confluent cells were sub-cultured every 3–4 days. For proliferation experiments, the cells were seeded in 96-well plates (TPP) at a density of 5,000 cells/well 24 h prior to the addition of each agent or their combinations. For flow cytometric analyses, the MCF-7 cells were seeded in 60-mm Petri dishes (TPP) at a density of 210,000 cells/well. For microscopic assessments, the MCF-7 cells were seeded in 12-well plates (TPP) at a density of 35,000 cells/well.

Cell lines K562 and MCF-7 were obtained from the European Collection of Cell Cultures. The cell lines were cultivated in a Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in 5% CO₂. For the viability assays, cells were seeded into 96-well plates (5000 cells per well), and after the preincubation period, were treated in triplicates with six different doses of each compound for 72 h. After treatments, a resazurin (Sigma-Aldrich, St Louis, MO, USA) solution was added for four hours, and the fluorescence of resorufin formed in live cells was measured at 544 nm/590 nm (excitation/emission) using a Fluoroskan Ascent microplate reader (Labsystems, Finland). The GI₅₀ value, a drug concentration lethal to 50% of the cells, was calculated from the dose–response curves.

The cytotoxicity assays were performed using the experimental procedure described in our previous research. Briefly, KB, HeLa, and MCF-7 cell lines were obtained from The European Collection of Cell Cultures (ECACC) supplied by Sigma-Aldrich (St. Louis, MO, USA). whereas A-549, U-87MG, and HDF cell lines were purchased from the American Type Cell Collection (ATCC) through LGC Standards. Approximately 0.1 mL of the diluted cell suspension (ca. 10,000 cells) was added to every well of the microtiter plate. A partial monolayer was formed after 24 h, and the supernatant was washed out. Then, 100 μL of 6 different 5-FU concentrations (0.1, 0.2, 1, 2, 10, and 20 μM) or number of MIPs that release the corresponding amount of 5-FU were added to the cells in microtiter plates. For NIPs blank experiments, the same mass of microparticles was used as for MIPs. Other experimental details were described in our previous work.