Progesterone pg

HC11 mouse mammary epithelial cells were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). HC11 cells were cultured at 37°C with 5% CO₂ in RPMI-1640 (Gibco, Grand Island, NY, United States) medium supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco), 5 μg/mL insulin (Sigma) and 10 ng/mL epithelial growth factor (EGF, Gibco). BMP4 (Abcam, Cambridge, MA, United States) is obtained from Escherichia coli, reconstituted in 10 mM citric acid and suitable for functional studies. BMP2 (PeproTech, Rocky Hill, NJ, United States) is obtained from Escherichia coli, reconstituted in deionized water containing 0.1% BSA and biological activity verification. For BMPs treatment, HC11 cells were serum starved overnight and then treated with BMP4 (50 ng/mL) or BMP2 (50 ng/mL) at the indicated time points. 10 mM citric acid was used as vehicle control for BMP4, deionized water containing 0.1% BSA was used as vehicle control for BMP2. For hormone stimulation, HC11 cells were grown to 70% confluence and serum starved overnight, then stimulated with 100% ethanol-dissolved 10 nM β-estradiol (E2, Sigma) and/or 100 nM progesterone (Pg, Sigma), 100% ethanol was used as vehicle control, the cells were harvested at 24 h after this treatment.

After several washes with sterile phosphate-buffered saline (PBS), chorionic villous explants from placental tissues (n = 4, placentae from first trimester of gestation; n = 10 placentae from term of gestation) were established as described by Caniggia with minor modifications [23 (link)]. Explants were incubated overnight at 37 °C in an atmosphere of 3% O₂, 5% CO₂ for first-trimester explants or 8% O₂, 5% CO₂ for term explants in DMEM F12 (phenol red and serum-free) plus L-glutamine (Gibco, Invitrogen, Basel, Switzerland and 1% antibiotics (penicillin-streptomycin). Medium was then replaced with a fresh one and placental explants cultured for 48 h.
n = 3 placentae from term of gestation were also used to establish placental explants for hormone exposure experiments. After the overnight incubation, term placental explants were treated with hormones according to their physiological levels found in the maternal serum at the end of gestation [24 (link)]: 1 × 10⁻⁸ M Estradiol (E2), 7 × 10⁻⁸ M Estriol (E3) and 1 × 10⁻⁶ M Progesterone (Pg) (Sigma Chemical Co. St. Louis, MO, USA) for 48 h.
At the end of experiments, explant cultures and their respective supernatants were collected and processed for protein analyses or subsequent analysis.

Progesterone (PG) was obtained from Sigma Aldrich (Dorset, UK). Hydroxypropyl methylcellulose acetate succinate (HPMCAS grade HF, average M_W 20,000 Da) was gifted by Shin-Etsu (Tokyo, Japan). Polyvinylpyrrolidone (PVP K30, average M_W 40,000 Da), silicon dioxide (SiO₂), hydroxypropyl methylcellulose (hypromellose, HPMC) (viscosity range of 3000–5600), microcrystalline cellulose (MCC) and deuterium oxide (D₂O) were obtained from Sigma Aldrich (Gillingham, UK). All other solvents were obtained from VWR International (Leicestershire, UK). All chemicals were of analytical grade and were used as received. Chemical structures of the experimental drug and polymers are presented in Figure 1.