Mek inhibitor

Three to four pseudopregnant day 4 mouse uterine horns were cut into small pieces (2–3 mm). Tissue pieces were first digested in 3 ml fresh medium (HBSS antibiotic; Gibco) containing 6 mg/ml dispase (Gibco) and 25 mg/ml pancreatin (Sigma), and then incubated in fresh medium containing 0.5 mg/ml collagenase (Sigma) at 37 °C for 30 min. The digested cells were passed through a 70 μm filter to obtain the stromal cells. Cells were plated at 60 mm dishes or 6-wells plates, containing phenol red-free Dulbecco modified Eagle medium (DMEM) and Ham F12 nutrient mixture (1:1) (Gibco) with 10% charcoal-stripped fetal bovine serum (CS-FBS) and antibiotic. Two hours later, the medium was replaced with fresh medium (DMEM/F12, 1:1) with 10% CS-FBS. The next morning, the medium was replaced with DMEM/F12 containing 1% C-FBS, E2 (Sigma, 10 nM), P4 (Sigma, 1 μM) and antibiotic to induce decidualization. The media was changed every 48 h. For the treatment of primary uterine stromal cells, MI-503 (2 μM; MedChemExpress), recombinant murine FGF2 (PeproTech, 20 ng/ml), recombinant mouse PTX3 (R&D systems, 100 ng/ml) and MEK inhibitor PD0325901 (Selleck, 5 μM) were used.