17β estradiol e2

Eight weeks old ICR mice were used in fertility test and other animal experiments. Cdo KO mice were generated by using 129 mice (Additional file 1). Mice were raised in controlled temperature (25 ± 1 °C) and humidity (60%–70%) with a 12 h light, 12 h dark cycle. The animal experiments were approved by the Chinese Association for Laboratory Animal Sciences. Virgin female mice were mated with sexually matured males to induce pregnancy (day 1 is the day of vaginal plug checked, d 1). Embryos were collected from oviducts on d 1. The implantation sites (IS) were visualized by intravenous injection of 0.1 mL 1% Chicago blue dye (Sangon Biotech, Shanghai, China) in saline on d 5 [39 (link)]. To investigate the ovarian hormonal influence on uterine CDO expression, wild-type (WT) mice were ovariectomized. Oil, 100 ng/mouse 17β-estradiol (E₂; MedChemExpress, NJ, USA) or 2 mg/mouse progesterone (P₄; MedChemExpress, NJ, USA) was injected 7 d later [40 (link)]. The treated mice were then sacrificed at indicated times for further experiments.

Several human cell lines were used in our study. GBC‐SD, RBE, Patu8988, AsPC1 and HIBEpiC were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). NOZ was provided by the Health Science Research Resources Bank (Osaka, Japan). QBC‐939 and SGC‐996 were obtained by the Academy of Life Sciences, Tong Ji University (Shanghai, China). GBC‐SD, Patu8988, AsPC1 and HIBEpiC were maintained in DMEM, RBE, SGC‐996 and QBC‐939 were maintained in RPMI‐1640, and NOZ was cultured in William's E medium (Gibco, NY, USA), containing 10% foetal bovine serum (FBS) and antibiotics (Gibco, NY, USA). Phenol red‐free DMEM and Charcoal Stripped Fetal Bovine Serum (Gibco, NY, USA) were used in E2 treatment experiments. Cells were maintained in controlled humidified atmosphere with 5% CO2 under 37°C. Tamoxifen, compound C, DCFDA, NAC and 17β‐estradiol (E2) were purchased from Medchem Express (MCE, USA). For inhibition of AMPK or ERɑ, cells with 65%‐70% confluence were transfected with shRNAs (AMPK: CAGGCCCAGAGGTAGATAT and AGAGAAATTCAGAACCTCA; ERɑ: GCCCTACTACCTGGAGAACGA and CTACAGGCCAAATTCAGATAA) or corresponding controls (GenePharma, Shanghai, China) by Lipofectamine 2000 (Invitrogen). Experiments were performed in accordance to the manufacturer's instructions. After 48 hours, the cells were collected and applied to subsequent experiments.