Total proteins were obtained from RL95-2 cells treated with or without 5, 10 or 20 µM PD for 24 h, or from ESCs by lysis using cold RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined using a BCA Protein Assay kit (Beijing Solarbio Science & Technology Co., Ltd.) and protein samples (20 µg per lane) were separated on 10% gels by SDS-PAGE (Thermo Fisher Scientific, Inc.), transferred onto PVDF membranes (MilliporeSigma) and incubated for 1 h at room temperature with 5% skimmed milk. The membranes were then incubated at 4°C overnight with the following primary antibodies: Ki67 (1:1,000; ab92742), proliferating cell nuclear antigen (PCNA; 1:1,000; ab92552), MMP2 (1:1,000; ab92536), MMP9 (1:1,000; ab76003), ADRA2A (1:1,000; ab85570), phospho (p)-PI3K (1:1,000; ab182651), p-Akt (1:1,000; ab192623), PI3K (1:1,000; ab191606), Akt (1:1,000; ab179463) and GAPDH (1:2,500; ab9485), all from Abcam. After washing with TBST (0.1% Tween), the membranes were incubated with Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (1:2,000; ab6721; Abcam) at 37°C for 1 h. The immunoreactive protein bands were visualized using an enhanced chemiluminescence detection system (Amersham; Cytiva) according to the manufacturer's instructions. Subsequently, protein brands were observed using ImageJ (v1.8.0; National Institutes of Health) and quantified by densitometry (QuantityOne 4.5.0 software; Bio-Rad Laboratories, Inc.). GAPDH served as an internal reference.