Spec 2

Two EC cell lines, Ishikawa (the estrogen-dependent EC cell line) and SPEC-2 (the non-estrogen-dependent EC cell line), were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained according to the provider’s instructions in DMEM/F12 (Gibco, Auckland, NZ) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA). All cells were grown until confluent and incubated in serum-free medium for 24 h before treatment with various experimental agents. Cross-linking with 3,3′-Dithiobis(sulfosuccinimidyl propionate) (DTSSP) was conducted to identify the interaction between AMF and GPER-1. Endometrial cells were washed with PBS and then exposed to DTSSP for 1 h at 4 °C, and the reaction was terminated by the addition of 20 mmol/L Tris–HCl (pH 7.5) for 15 min at room temperature (RT). Cells were extracted with lysis buffer, and insoluble material was removed by centrifugation. Supernatants were then processed for coimmunoprecipitation. Exogenous AMF/PGI was diluted to 10 ng/ml with PBS to stimulate EC cells.