Dulbecco s modified eagle s medium dmem ham s f 12 medium

Ishikawa cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). The medium was replenished every 48 h. The human endometrial stromal cell lines (CRL-4003) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium (Sigma-Aldrich, Saint Louis, MO, United States) supplemented with 10% charcoal-stripped FBS (Biological Industries, Israel) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). All cells were cultured in a 5% CO₂ incubator at 37°C. The circ-CYP24A1 siRNA, miR-224 mimic, miR-224 inhibitor, and PRLR siRNA were transfected using Lipofectamine 2000 (Lip 2000; Invitrogen, Carlsbad, CA, United States). The cells were cultured for 2 h in a penicillin/streptomycin-free medium before transfection. SiRNA and Lip 2000 were diluted separately in 50 μl RPMI-1640 or DMEM/Ham’s F-12 medium and mixed for 15 min at 25°C away from light. Equal amounts of components were then added to orifice plates, and the penicillin/streptomycin medium was added to make the total volume of 500 μl. The circ-CYP24A1, PRLR, and negative control (NC) siRNAs were purchased from Sangon Biotech (Shanghai, China). The miR-224 mimic, miR-224 inhibitor, and NC were purchased from RuiBo Biotechnology (Guangzhou, China).

Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium (1:1) (with and without phenol red), phosphate-buffered saline (PBS) Ca²⁺/Mg²⁺-free, bovine serum albumin (BSA) fraction V, glutamine, antibiotics, collagenase type IV, NaOH, Bradford reagent, 4′,6-Diamidino-2-phenylindole (DAPI), hydrogen peroxide, sildenafil citrate salts (98%), propidium iodide, ribonuclease enzyme, a redox-sensitive probe, and all reagents for Western blotting were from Sigma Aldrich (St. Louis, MO, USA). Fetal calf serum was from Gibco^® (Grand Island, NY, USA). 2-mercaptoethanol was from Life Technologies, Inc. Laboratories (Grand Island, NY, USA). Cy3-labeled secondary antibodies (Abs) were from Jackson Laboratory (Bar Harbor, ME, USA); peroxidase secondary Abs and all reagents for Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) were from Millipore (Billerica, MA, USA). Plastic ware for cell cultures and disposable filtration units for growth media preparation were purchased from Corning (Milan, Italy).