Nobiletin

At approximately 21 hpi, a total of 7237 (3398 for MN_EGA phase and 3839 for MJ_EGA phase) presumptive zygotes were denuded of cumulus cells (CCs) by vortexing for 3 min and then cultured in groups of 50 in a four-well dish containing 500 µL per well of culture medium (synthetic oviductal fluid (SOF);^{57 (link)} supplemented with 5% (v/v) FCS, 4.2 mM sodium lactate, 0.73 mM sodium pyruvate, 30 µL/mL basal medium eagles (BME) amino acids, 10 µL/mL minimum essential medium (MEM) amino acids and 1 µg/mL phenol red, in the presence (MN_EGA) or absence (MJ_EGA) of nobiletin (MedChemExpress, MCE, Sweden) or with 0.03% dimethyl sulfoxide (DMSO vehicle for nobiletin dilution). At 54 hpi those embryos that reached the 8-cell stage were selected and randomly cultured in groups of 50 in SOF + 5% FCS until Day 8 (MN_EGA) or in presence of nobiletin or DMSO until 96 hpi (MJ_EGA). At 96 hpi those embryos that reached the 16-cell stage were selected and randomly cultured in groups of 50 in SOF + 5% FCS until Day 8 (MJ_EGA) (see ‘Experimental design’ described below for more details). Culture took place at 38.5 °C in an atmosphere of 5% CO₂, 5% O_2, and 90% N₂.

Nobiletin (LCMS purified, 99.04%; dissolved in dimethyl sulfoxide, final dose 0.0005% v/v) was obtained from MedChemExpress (Jersey City, NJ, USA). The reagents and materials for each kit are listed with each method.

Nobiletin (>99% purity) was purchased from MedChemExpress (Monmouth Junction, NY, USA); the CCK-8 solution was obtained from Dojindo Molecular Technologies, Inc. (Tokyo, Japan); fetal bovine serum was obtained from ZETA (San Francisco, CA, USA); trypsin and dimethyl sulfoxide (DMSO) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); Eagle’s minimum essential medium (EMEM) and McCoy’s 5A media were obtained from Gibco (Gaithersburg, MD, USA). Antibodies against AKT, STAT3, SRC, YY1AP1, caspase 3, caspase 9, phospho-AKT, phospho-STAT3, phospho-YY1AP1, phospho-SRC, cleaved caspase 3, and cleaved caspase 9, as well as all secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). Stattic and Verteporfin were obtained from MedChemExpress. IGF1 was obtained from BD Bioscience (Shanghai, China).