Stable glutamine

MCF-7 cells were chosen for their hormone receptor-positive attributes, reflecting the luminal breast cancer subtype. Their estrogen and progesterone receptor presence aligns with our hormone-related focus. Our selection was guided by their relevance to our objectives, well-documented status, and specific interest in MTE's impact on this hormone receptor-positive subtype. Additionally, our choice of MCF-7 cells was influenced by the existing data on green tea phenol's interaction with ERα, while information about ERβ interactions remains limited.
MCF-7 cells were cultured on an 80% monolayer in a cell culture bottle. Dulbecco's modified Eagle medium (DMEM; 3.7 g/l NaHCO3, 4.5 g/l d-glucose, 1.028 g/l stable glutamine, and sodium pyruvate; Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS; Biochrom, Berlin, Germany) was used for cultivation. The cells were incubated with atmospheric concentrations of CO₂ of 5% at 37 °C and were then trypsinized and counted for further use.

gp91^phox−/− mice were a kind gift from Dr. Katrin Breitbach (Friedrich Loeffler Institute of Medical Microbiology, Ernst-Moritz-Arndt University, Greifswald). Wild type control C57BL/6J^Bom mice were obtained from Taconics. Mice were kept in pathogen-free conditions (SPF). Spleens from mice were passed through a fine mesh filter (BD Falcon) to obtain a single-cell suspension. T cells were purified by non-T cell depletion using mouse pan T-cell isolation kit and AutoMACS (all from Miltenyi Biotec). Purity of T cells, determined by flow cytometry, was routinely more than 96%. The cells were cultured at 10⁶ cells/ml, 37°C and 5% CO₂ in RPMI 1640 medium supplemented with stable glutamine (Biochrom AG), 10% fetal calf serum PAN Biotech), 2 μg/ml ciprobay (Bayer) and 50 μM β-mercaptoethanol (Sigma Aldrich).

The cell lines MCF7 and T47D were used as models for the ductal breast carcinoma. The cells were cultured in DMEM (3.7 g/L NaHCO3, 4.5 g/L D-glucose, 1.028 g/L stable glutamine, and Na-Pyruvate; Biochrom, Berlin, Germany). After adding 10% heat-inactivated fetal calf serum (FCS; Biochrom) to the medium the solution was incubated at an atmospheric CO₂ concentration of 5% and at 37 °C in an incubator to lyse.
MCF7 and T47D cells were separately grown in sterile 12 multi-well slides at a density of 500,000 cells/mL DMEM with 10% FCS. The medium was changed after 4 h to pure DMEM without any FCS. After 20 h, the cells were stimulated with 1 μM epinephrine (Sigma-Aldrich Chemie GmbH, Munich, Germany). The cells were stimulated for 6 h with 10 μM epinephrine in the case of Western blot samples. Control cells were incubated in parallel without any stimulants.

We used IPEC-J2 cells (DSMZ, Braunschweig, Germany), a non-transformed cell line that stems from porcine jejunal epithelia, to analyze whether the changes in epithelial barrier function in PP tissue also occur in a cell culture model. Cells were seeded on semi-permeable cell culture inserts (Millipore, Darmstadt, Germany) at 10⁵ cells per filter. Dulbecco’s MEM/Ham’s F12 with 3.15 g/L glucose and 2 mM stable glutamine (Biochrom, Berlin, Germany) was used and supplemented with 10% porcine serum and 1% penicillin/streptomycin (Sigma Aldrich, Munich, Germany). Medium was changed every 2–3 days, and filters were filled with 500 μl apically and 1 ml basolaterally. By using a chopstick electrode and an epithelial Volt/Ohm Meter (EVOM, World Precision Instruments, Sarasota, FL, United States), we measured the TER immediately before each media exchange, and values were corrected with the media and blank values used in our experimental settings. Once the cells had built up a confluent monolayer, resulting in consistent TER values, the incubation experiments were started. We added 5000 U/ml TNF (PeproTech, Hamburg, Germany) to the basolateral side of the cell filters, and TER was recorded for up to 10 h. Cell passages between 8 and 13 were used for experiments.

To mimic ductal breast carcinoma, the T47D cell line was used. Cells were grown on 80% monolayer in a cell culture bottle and cultivated in Dulbecco's Modified Eagle Medium (DMEM; 3.7 g/L NaHCO3, 4.5 g/L d-glucose, 1.028 g/L stable glutamine, and sodium pyruvate; Biochrom, Berlin, Germany). Then 10% heat-inactivated fetal calf serum (FCS; Biochrom) was added to the medium and incubated with atmospheric concentrations of CO₂ of 5% at 37 °C. For further use they were trypsinized and counted.