Crl 1474

Normal human skin fibroblasts (CRL-1474) were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were grown in the culture medium DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin at 37 °C in a 5% CO₂ incubator. For the experiments, the culture medium was replaced with the serum-free medium. MP, PP, and RA were dissolved in 99.5% ethanol and stored as the concentrated solutions at 4 °C. For the experiments, they were appropriately diluted in DMEM and added to the cells in such volumes that the final concentration of ethanol did not exceed 0.1%. In each experiment, fibroblasts were incubated for 24 h in the serum-free culture medium in the presence of parabens (MP and PP) alone, parabens combined with RA, and RA alone, and an appropriate amount of the solvent (ethanol), and the latter constituted the controls. The tested compounds were used at the following concentrations: parabens (0.001% MP and 0.0003% PP, 0.003% MP and 0.001% PP, and 0.01% MP and 0.003% PP), and RA: 50, 100, 150 µM.

The study was performed on the normal human skin fibroblast line (CRL-1474) purchased from American Type Culture Collection (Manassas, VA, USA). Fibroblasts were cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) as well as 2 mM glutamine (Quality Biologicals Inc., Gaithersburg, MD, USA), penicillin (50 U/mL) (Quality Biologicals Inc., Gaithersburg, MD, USA), and streptomycin (50 µg/mL) (Quality Biologicals Inc., Gaithersburg, MD, USA) at 37 °C in a humidified incubator in atmosphere containing 5% CO₂. For experiments fibroblasts were grown to 90% confluence and the cultured medium was changed to DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) without serum before addition of compounds. BP-3 (Sigma-Aldrich Corp., St. Louis, MO, USA) and RA (BIOKOM, Warsaw, Poland) were dissolved in DMSO (Sigma-Aldrich Corp., St. Louis, MO, USA) and stored as the concentrated solutions at 4 °C. Fresh dilutions using DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) were prepared just before adding the reagents to the cells with the final DMSO (Sigma-Aldrich Corp., St. Louis, MO, USA) concentration not exceeding 0.1% (v/v).

All studies were performed on normal human skin fibroblasts (CRL-1474), which were purchased from American Type Culture Collection, Manassas, VA, USA. The cells were maintained in DMEM supplemented with 10 % fetal bovine serum (FBS), 2 mmol/l glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37 °C in a 5 % CO₂ incubator. Cells were counted in hemocytometer and cultured at 1 × 10⁵ cells per well in 2 ml of growth medium in six-well plates (Costar). Cells reached confluence at day 6, and in most cases, such cells were used for assays. Cells were used in the 8th to 14th passages.

Human breast cancer cell line MDA-MB-231 and human skin fibroblasts (CRL1474) were obtained from American Type Culture Collection (ATCC). Cells were maintained in high-glucose DMEM supplemented with 10% heat-inactivated foetal bovine serum GOLD (FBS GOLD), 2 mM l-glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were cultured in Falcon flasks (BD) in a 5 % CO₂ incubator (Galaxy S+; New Brunswick), at 37 °C. Subconfluent cultures were detached with 0.05 % trypsin and 0.02 % EDTA in calcium-free phosphate-buffered saline (PBS) and counted in a Scepter cell counter (Millipore).

Human skin fibroblasts (CRL-1474) were obtained from the American Type Culture Collection (ATCC). Sterile reagents for cell culture, including AlgiMatrix 3D Cell Culture System, were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Phospholipid and ceramide internal standards were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All solvents were of LC-MS grade. Milli-Q water was used for all experiments, filtered through a 0.22 µm filter and obtained using a Milli-Q Millipore system (Advantage A10, Millipore Corporation, Billerica, MA, USA).

All studies were performed on normal human skin fibroblasts (CRL-1474), that were purchased from American Type Culture Collection, Manassas, VA, USA. The cells were maintained in DMEM supplemented with 10 % fetal bovine serum (FBS), 2 mmol/l glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37 °C in a 5 % CO₂ incubator. Cells were counted in hemocytometer and cultured at 1 × 10⁵ cells per well in 2 ml of growth medium in 6-well plates (Costar). Cells reached confluence at day 6 and in most cases such cells were used for assays. Cells were used in the 8th to 14th passages.