Etonogestrel (Implanon) is a steroidal progestin used as hormonal contraceptives.

Etonogestrel induces FKBP51 mRNA and protein expression in cultured HESCs[1].

First, 0.5 mL tissue homogenate is diluted with 1 mL water. Then, to this mixture, 2.5 mL ethanol and 1.5 mL chloroform (all reagents chilled) are added and shaken for 1 min at 4°C, then centrifuged. The enzyme activity in the supernatant is determined. The assay mixture contained 1.2 mL sodium pyrophosphate buffer (0.025 M, pH 8.3), 0.1 mL 186 mM phenazine methosulfate (PMS), 0.3 mL 30 mM Nitroblue tetrazolium (NBT), and 0.2 mL of nicotinamide adenine dinucleotide (NADH), and appropriately diluted enzyme preparation and water in a total volume of 3 mL. Reaction is initiated by the addition of NADH. After incubation at 30°C for 90 min, the reaction is stopped by the addition of 1 mL glacial acetic acid. The reaction mixture is stirred vigorously and shaken with 4 mL n-butanol. The intensity of the chromogen in the butanol layer is measured at 560 nm against a butanol blank. A system without enzyme served as control. One unit of enzyme activity is defined as 50% inhibition of NBT reduction in 1 min under the assay conditions.

Thawed HESCs are grown to confluence in basal medium (BM), a phenol red-free 1:1 v/v mix of Dulbecco's MEM/Ham's F-12, with 100U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone complex supplemented with 10% charcoal-stripped calf serum. Confluent HESCs are incubated in parallel in BM with 0.1% ethanol (vehicle control) or 10 nM E2(estradiol) or E2+100 nM P4(progesterone), or 100 nM of ETO(etonogestrel) or 100 nM of MPA(medroxyprogesterone acetate). After 7 days, the cultures are washed twice with 1X phosphate-buffered saline (PBS) to remove residual serum, then placed in a serum-free defined media (DM) comprising BM plus ITS+ (insulin, transferrin, selenious and linoleic acid) premix, 5 μM of FeSO4, 50 μM of ZnSO4, 1 nM of CuSO4, 20 nM of Na2SeO3 trace elements, 50 μg/ml of ascorbic acid and 50 ng/ml of recombinant epidermal growth factor for 24h. The cultures are washed twice with 1X PBS to remove the residual steroids and DM containing corresponding vehicle control or steroids is added to the cultures for 6h or 24h. At the end of incubation periods, HESCs are washed with ice-cold 1X PBS and stored at ?70°C until used for total RNA and protein extraction.(Only for Reference)