Verteporfin

To explore the effect of YAP-mTOR signal on the decidualization of the eutopic ESCs, we performed an in vitro decidualization induction of the eutopic ESCs after interfering with the YAP function by YAP-TEAD inhibitor Verteporfin (S1786, Selleckchem, USA) (1 μM, 18 h) and blocking the mTOR signal by Rapamycin (S1039, Selleckchem, USA) (100 nM, 4 h) which also can induce autophagy. Verteporfin and Rapamycin treatments were described previously (24 (link)). DMSO is the negative control group. Decidualization was induced in ESCs at 70% to 80% confluence after Verteporfin and Rapamycin treatments in 10% charcoal-stripped phenol red-free medium for 24 h. The medium was then replaced, and the cells were cultured with 2% charcoal-stripped phenol red-free medium supplemented with 0.5 mM 8-Br-cAMP (Abcam, Cambridge, MA) and 1 mM medroxyprogesterone acetate (MPA) (Sigma-Aldrich, St. Louis, MO) for 72 h. The culture medium was collected every 24 h, and the supernatants were incubated at −20°C to detect decidual prolactin (dPRL). dPRL protein levels in the supernatants, which are a representative marker of decidual cells, were determined using a commercially available ELISA kit (Cusabio Biotech, Wuhan, China). The ESCs were observed under an inverted microscope every 24 h to study their morphological changes.