Fisetin

Astragaline (CAS No. : 480-10-4, purity of 99.85%), Afzelin (CAS No. : 482-39-3, purity of 99.62%), Quercetin 3-O-α-L-arabinoside (CAS No. : 22688-79-5, purity of 99.83%), Narcissoside (CAS No. : 604-80-8), Avicularin (CAS No. : 572-30-5), Fisetin (CAS No. : 528-48-3, purity of 98.02%), Herbacetin (CAS No. : 527-95-7, purity > 99.0%) were purchased from MedChemExpress (MCE, Shanghai, China). 3'-Hydroxyflavanone (CAS No. : 1621-55-2), Kaempferol-3-O-rutinoside (CAS No. : 17650-84-9, purity ≥ 98%) were obtained from YuanYe Biotechnology (Shanghai, China).

MII oocytes were collected from 6–8-week-old female mice, as described previously [17 (link)]. Briefly, the mice were injected intraperitoneally with 5–10 IU PMSG, followed by 5–10 IU hCG 46–48 h later to induce oocyte aging. The MII oocytes were retrieved from the ampulla 16 h later and collected in M2 medium (Sigma, St. Louis, MO, USA). The oocytes with surrounding cumulus cells (COCs) were cultured in 500 μL M16 medium (Sigma) in a Nunc™/SPL IVF 4-well dish (ThermoFisher Scientific, Waltham, MA, USA) for 12 h in humid air (37 °C, 5% CO₂). Fisetin (MedChemExpress, Shanghai, China) was dissolved in DMSO (Sigma) at 80 mM and stored at −80 °C. The optimal Fisetin concentration was determined using dose–response gradients. The same volume of DMSO was added into the control groups. The final concentrations did not exceed 1%.

COV434 cells and primary pT&S cells were transfected with siRNA (RIBOBIO) targeting FOXP1, SOX4 and FOS using Lipofectamine 3000 (Thermo, L3000015). The sequences of the siRNAs are listed in Supplementary Table 5. In brief, cells were incubated in 50 nM siRNAs for 6 h and cultured in DMEM containing 10% FBS for another 48 h. For exploring senolytics fighting ovarian cell senescence, cells were treated with quercetin (10 μM, MedChemExpress), dasatinib (100 nM, MedChemExpress) or fisetin (10 μM, MedChemExpress) for 24 h. After that, cells were used for RT–qPCR, western blot, SA-β-gal staining and immunofluorescence.