BeWo and JAR cells were purchased from the European Collection of Cell Cultures (ECACC) and were cultured as previously described [18 (link)]. In brief, BeWo were cultured in DMEM/F12 (1:1, Gibco, life technologies; Paisley, UK) supplemented with 10% FCS (Gibco), penicillin/streptomycin (Gibco), amphotericin B (Gibco) and l -glutamine (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C. JAR cells were cultured in RPMI medium (Gibco) including same supplements and culture conditions as described for BeWo. Cells between passage 10 and 20 were used for in vitro experiments. In case of forskolin treatment, culture medium was supplemented with forskolin (Tocris, Bio-techne, Abingdon, UK) at a final concentration of 20 µM (20 mM stock in DMSO). Control cells were incubated with culture medium containing the same volume of DMSO (0.1%).
For leupeptin treatments, BeWo cells (2 × 105 cells/well) were plated in 12-well dishes (Nunc Lab-Tek, Thermo Fisher; NY, USA) in above described culture medium 1 day prior experimental start. Next day, cells were incubated in the presence or absence of forskolin (20 µM) for 48 h at 37 °C. leupeptin (Sigma-Aldrich, St. Louis, MO, USA) was added to cultures 24 h prior to the end of experiments at a concentration of 100 µM. DMSO (0.1%) and aqua dest were used as vehicle controls for forskolin and leupeptin, respectively.
For leupeptin treatments, BeWo cells (2 × 105 cells/well) were plated in 12-well dishes (Nunc Lab-Tek, Thermo Fisher; NY, USA) in above described culture medium 1 day prior experimental start. Next day, cells were incubated in the presence or absence of forskolin (20 µM) for 48 h at 37 °C. leupeptin (Sigma-Aldrich, St. Louis, MO, USA) was added to cultures 24 h prior to the end of experiments at a concentration of 100 µM. DMSO (0.1%) and aqua dest were used as vehicle controls for forskolin and leupeptin, respectively.