Medroxyprogesterone acetate

Methanol (≥99.9%) and acetonitrile (≥99.9%) were purchased from Sigma-Aldrich (St. Louis, USA). All reagents used were of analytical grade. Anhydrous magnesium sulfate (MgSO₄), sodium chloride (NaCl), primary secondary amine (PSA), and alumina-N were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). High-purity (least 98% purity) analytical standards were used. Norethisterone, 17α-hydroxyprogesterone, D-methylnorethindrone, 17α-hydroxyprogesterone acetate, megestrol, medroxyprogesterone, chlorprogestone acetate, medroxyprogesterone acetate, melengestrol acetate, and progesterone were purchased from Sigma-Aldrich (St. Louis, USA).
Individual stock standard solutions (1000 mg/L) of each compound were prepared using Methanol and stored in the refrigerator at −20°C. The mixed standard solution was prepared by mixing the 10 individual stock standard solutions (100 μL for each) and diluting with Methanol to 10 mL in a volumetric flask, and the final concentration of each progestin was 10 mg/L. The mixed standard solutions prepared were stored at 4°C.
Acquity ultra-performance liquid chromatography (Waters USA); Synapt G2-Si quadrupole time-of-flight mass spectrometry (Waters USA); GR22GIII high-speed refrigerated centrifuge (HITACHI Japan); N-EVAPTM 112 nitrogen blowing apparatus (Organomation, USA); and KQ-500DE numerical control ultrasonic cleaner (Kunshan, China).

SN33638 was synthesized as described previously (35 (link)). Androstenedione, estrone, indomethacin, flufenamic acid, medroxyprogesterone acetate, and apigenin, were purchased from Sigma-Aldrich, 5-(3-bromo-4-hydroxybenzylidene)-3-(4-methoxyphenyl)-2-thioxothiazolidin-4-1 (17β-HSD3 inhibitor) was purchased from Merck Millipore and PGD₂ was purchased from Cayman Chemical Company.

K562 cells were cultured and polar cell extracts were prepared as described before [4 (link)]. For tracer-based metabolic analysis, standard glucose- or glutamine-free RPMI-1640 media (Gibco) was used and substituted with 2 g/l [1,2-¹³C]glucose or 300 mg/l [3-¹³C]glutamine (Isotec, Sigma), respectively. 5 × 10⁷ exponentially growing K562 cells per control or treatment were pelleted by centrifugation at 8000g for 5 min and resuspended in the relevant media with BaP or solvent controls and incubated for 24 or 3 h at 37 °C and 5 % CO₂ in a humidified incubator. For BaP treatment, bezafibrate 0.5 mM and medroxyprogesterone acetate 5 μM (Sigma) or the equivalent concentrations of DMSO and ethanol solvent control were added to the media at T = 0 h.
For 3-h labelling of 24-h drug treatments, media was exchanged for the last 3 h with media supplemented with labelled glucose or glutamine plus BaP or solvent control. All samples were dissolved in 100 mM phosphate buffer with 10 % D₂O and 500 μM Trimethylsilylpropanoic acid (TMSP) added.

Human EnSC were isolated from endometrial biopsies as described previously.²⁸ Briefly, endometrial biopsies were subjected to enzymatic digestion using 500 µg/mL of collagenase type Ia (Sigma‐Aldrich, Poole, UK) and 100 µg/mL of DNase I (Lorne Laboratories Ltd, Reading, UK) for 1 hour at 37°C. Digested tissue was filtered through a 40 µM cell strainer to remove glandular cell clumps, and the flow‐through collected and cultured in DMEM/F12 (Thermo Scientific, Loughborough, UK) containing 10% dextran‐coated charcoal‐treated fetal bovine serum (DCC‐FBS), 1 × antibiotic‐antimycotic mix, 10 µM of L‐glutamine (Thermo Scientific), 1 nM of estradiol, and 2 μg/mL of insulin (Sigma‐Aldrich). Cells were lifted with 0.05% trypsin and re‐seeded as required. To induce decidual transformation, confluent EnSC monolayers were downregulated in phenol‐free DMEM/F‐12 media containing 2% DCC‐FBS and decidualized with 10 µM of medroxyprogesterone acetate (MPA) and 0.5 mM of 8‐bromo‐cAMP (C+M treatment) (Sigma‐Aldrich). To study the effect of exogenous HA, decidualizing EnSC were treated with either 100 µg/mL of HMWHA (molecular mass ~1320 kDa) or LMWHA (molecular mass ~33.0 kDa), purchased from Bio‐Techne (USA), as described elsewhere.²⁹, ³⁰ Medium was refreshed every 2 days. All experiments were performed at passage 2.