β oestradiol e2

5×10⁵ T47D cells were plated in 6-well dishes and cultured in RPMI-1640 and 10% HI-FBS for 24 h. The next day, the cells were washed with 1× PBS and the media was replaced with RPMI-1640 (phenol red free) (Thermo Fisher) and 10% Dextran Coated Charcoal stripped (DCC) FBS for 48 h prior to steroid treatment. The cells were treated with either 10 nM β-oestradiol (E2) (Sigma), 10 nM progesterone (P4) (Sigma), 10 nM dihydrotestosterone (DHT) (Selleck Chemicals) or vehicle (100% EtOH) for the indicated times and then harvested. Cells were stored at −80 °C until analysed.

For the differentiation of both stromal-only and EO-only scaffolds, 10 nM β-oestradiol (E2, Sigma) was added 2 days post-cell seeding. For stromal cells only, at days 4 and 6, medium was replaced with the following conditions: 10 nM E2 + 1 μM progesterone (P4, Sigma) + 1 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP, Sigma). For EO-only scaffolds, 20 ng ml⁻¹ prolactin (PRL, Peprotech, UK) was added in addition at days 4 and 6 to mimic signals from decidualized stromal cells. Supernatant was collected at days 2, 4, 6 and 8 and frozen at −20°C for storage before downstream analysis.
Enzyme-linked immunosorbent assays (ELISAs) were used to measure protein concentrations of key markers of stromal decidualization (prolactin, a hormone secreted by decidual stromal cells) and epithelial differentiation (glycodelin, one of the principal components of the endometrial glands) in the supernatant. ELISAs for prolactin (DY682, R&Dsystems, USA) and glycodelin (ELH-PP14, Raybiotech, USA) were measured from stromal-only and EO-only scaffolds, respectively. A volume of 100 μl of supernatant was used with each sample measured in duplicate, as per the manufacturer's instructions.