Immortalized non-neoplastic human endometrial stromal cells (T HESCs) were cultured in the DMEM/F-12 medium without phenol red (Sigma, USA), supplemented with 10% fetal bovine serum (FBS; GIBCO, USA), 1% insulin transferrin selenium (ITS) + Premix (Corning 3454352, USA), and 500 ng/mL puromycin (InvivoGen, USA). Cells were maintained at 37 ℃ in a humidified atmosphere with 5% CO2.
For in vitro decidualization, T HESCs were maintained for 6 days in the DMEM/F-12 medium containing 1 μM medroxyprogesterone 17-acetate (MPA; Sigma) and 0.5 mM 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; Sigma), 2% FBS, and 1% ITS + Premix. The culture medium was replaced with a fresh medium every 2 days.
NOX4 siRNA, the miR-375 mimic, and the miR-375 inhibitor were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA) into T HESCs, following the manufacturer’s recommended protocol.
For in vitro decidualization, T HESCs were maintained for 6 days in the DMEM/F-12 medium containing 1 μM medroxyprogesterone 17-acetate (MPA; Sigma) and 0.5 mM 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; Sigma), 2% FBS, and 1% ITS + Premix. The culture medium was replaced with a fresh medium every 2 days.
NOX4 siRNA, the miR-375 mimic, and the miR-375 inhibitor were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA) into T HESCs, following the manufacturer’s recommended protocol.