Cell apoptosis

Cell apoptosis was analyzed using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Beijing Solarbio Science & Technology Co., Ltd.). The trophoblasts were first seeded in a 6-well plate (5×10⁴ cells/well) for 24 h, and then treated with PBS, siRNA-ANXA1 or empty vector using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at 37°C for further 24 h. The cells were digested by trypsin (Beijing Solarbio Science & Technology Co., Ltd.) and re-suspended with a 1X binding buffer. Next, the cells were double-stained with Annexin V-FITC/PI for 20 min, and the cell apoptosis was determined using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).