Penicillin g

BALB/c 3T3 clone A31-1-1 cells from the laboratory of M. Umeda (Hatano Research Institute, Japan) were kindly provided by Dr. A. Poth (Knoell Germany GmbH, Mannheim, Germany). The cells were cultured in minimum essential medium (MEM) supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM (v/v) l-glutamine, 100 µg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin (all components were obtained from Biochrom [Merck], Berlin, Germany). Chinese hamster ovary cells (subclone K1, CHO-K1-BH4) were kindly provided by B. J. Phillips in the context of the International Food Irradiation Project (IFIP; Ministry of Agriculture, Fishery and Food, UK 1984) and cultured in McCoy’s 5A medium supplemented with 10% (v/v) FCS, 100 IU ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin (all components were obtained from Life Technologies, Darmstadt, Germany). The human colon adenocarcinoma cell line Caco-2 was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany). These cells were cultured in MEM Eagle’s (EMEM) medium supplemented with 1% (v/v) non-essential amino acids, 2 mM (v/v) l-glutamine, 50 IU ml⁻¹ penicillin G, 50 µg ml⁻¹ streptomycin (all components were obtained from Lonza, Verviers, Belgium) and 10% (v/v) FCS obtained from Biowest (Nuaille, France). All cell lines were kept at 37 °C in a humidified atmosphere containing 5% CO₂.

BM-hMSCs were cultured as previously described [28 (link)]. Briefly, cells were seeded on a cell culture-treated surface (Corning, New York, USA) in the presence of Minimum Essential Medium (MEMα) manufactured under GMP conditions (Macopharma) and supplemented with either MSC-qualified FBS (Gibco, Life Technologies, Carlsbad, USA) with 1 ng/mL bFGF (Eurobio, Montpellier, France) or hPL or PR-hPL. Heparin (Biochrom, VWR, Radnor, USA) at 2 IU/mL was added to hPL- and PR-hPL-containing media to avoid gelation of the medium. 100 U/mL penicillin G / 0.1 mg/mL streptomycin sulfate (Lonza, Basel, Switzerland) was added under all conditions, and the media were renewed twice a week. Cell cultures were maintained in a humidified atmosphere containing 5% CO₂. All experiments were performed between P1 and P4.

The synthesis and characterization methods of the compound were published by Deniz et al. (2015). A solution of 25 mM was prepared in dimethyl sulfoxide (DMSO) as a stock solution. Further dilutions were made in a culture medium containing 0.1% DMSO. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with penicillin G (100 U/mL), streptomycin (100 µg/mL), L-glutamine (Lonza, Switzerland), and 10% fetal bovine serum (GIBCO, USA) at 37 °C in a humidified atmosphere containing 5% CO2.

Human vulvar SK-LMS-1 cells were purchased from ATCC (Manassas, VA, USA) and cultured in DMEM (Lonza, Cologne GmbH, Germany) supplemented with 10% foetal bovine serum (FBS; Sigma-Aldrich, Milan, Italy), 2 mM L-Glutamine (Lonza) and antibiotics (100 U of penicillin G and 100 μg/ml of streptomycin sulfate, Lonza). Cell cultures were maintained at 37°C under a humidified 5% CO₂ atmosphere. Conditioned medium (CM) was prepared from confluent SK-LMS-1 cells culture maintained in DMEM without serum. After 72 hrs the CM was collected, centrifuged at 500 × g for 5 min. at 4°C and filtered through a 0.22 μm pore size membrane.