Mcf10a

MDA-MB-231 (Sigma Aldrich, Vienna, Austria), HaCat (ATCC, US), MCF10A (LGC Promochem, US), A375 (ATCC, US), SW-872 (ATCC, US), 93T449 (ATCC, US), SW1353 (CLS, Germany) and juvenile fibroblasts fresh established from foreskin samples were obtained from Division of Biomedical Research (BMF), Medical University of Graz, Austria. HeLa (ATCC®, Guernsey, UK), fibroblast, HaCat, SW1353 and A375 cells were cultured in DMEM supplemented with 2 mM glutamine, 1% PS (100 U/mL penicillin, 100 μg/mL streptomycin) and 10% fetal bovine serum (FBS). MCF10A were cultured in DMEM with single quot kit suppl. Gr, 5% Horse Serum, 20 ng/mL hEGF, 0.5 μg/ml hydrocortison, 100 ng/ml choleratoxin, 10 μg/ml insulin and 2 mM glutamine. MDA-MB-231 were maintained in DMEM Hams F12 with 10% FBS, 2 mM glutamine and 1% PS. SW872 were cultured in DMEM Hams F12 supplemented with 5% FBS, 2 mM glutamine and 1% PS. 93T449 were cultured with RPMI-1640 with 10% FBS, 2 mM glutamine, 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1mM sodium pyruvate and 1% PS.
Cells were maintained in a humidified incubator at 37°C with 5% CO₂. HeLa cells were treated for up to 3 days with AdOx (40 μM), MS023 (10 μM), GSK715 (2 μM), GSK591 (1 μM) and DMSO, before cell extracts were prepared.

The MCF-10A breast epithelial cells (ATCC cat#CRL-10317, RRID:CVCL_0598; LGC Standards, Barcelona, Spain) were cultured in a DMEM/F12 culture medium and supplemented with the following compounds: 20 ng/mL epidermal growth factor; 2 mM of stable L-glutamine, 3.5 μg/mL human insulin, 0.5 μg/mL hydrocortisone, 10% heat-inactivated FBS, and 1% of a solution composed of penicillin/streptomycin. Cells were maintained in a humidified environment comprising 95% air and 5% CO₂, at a temperature of 37 °C. Cell subculturing was performed twice a week using trypsinization (0.25% trypsin/0.025% EDTA), ensuring that cells were maintained below a 90% confluence. Regular mycoplasma contamination tests were conducted on the cells.
Prior to each experimental assay, MCF-10A cells underwent trypsinization and were centrifuged at 457× g for five minutes at 20 °C. Cells were seeded onto 96- or 6-well plates (TPP, Biotecnómica) at various densities, according to the experimental assay. Depending on the experimental objectives, cells were exposed to isoprenaline (0.1–10 μM), adrenaline (0.1–10 μM), salbutamol (0.1–10 μM), UK 14,304 (0.1–10 μM), or propranolol (10 μM), either individually or in combination, and then incubated for up to 72 h. Control experiments included cells treated with the vehicle alone (0.1% DMSO) in each assay.