Ovarian Granulosa Cells Response to D-Galactose

The ovarian granulosa cells (KGN cell line; Procell) were cultured in DMEM (Sigma-Aldrich) added with 10% FBS (Sigma- Aldrich) and 1% antibiotic/antifungal solution (Invitrogen, Carlsbad, CA, USA). KGN cells in the 2^nd generation were put in 6-well plates at a density of 1×10⁵ cells/well. After 24 h, the original medium was discarded, and cells were only treated with DMEM/F12 medium (Invitrogen) for 24 h to starve the cells. Thereafter, KGN cells were assigned into 6 groups: blank (without treatment), D-gal (medium was added with 100 nM D-gal^{35 (link)}), D-gal + EVs (medium was supplemented with 100 nM D-gal and hucMSC-EVs at a density of 1×10⁸ particles/mL), D-gal + GW4869 (the CM was added with 100 nM D-gal and equivalent GW4869), D-gal + EVs + LY294002 (medium was supplemented with 100 nM D-gal, an equivalent volume of hucMSC-EVs, and 50 μM PI3K/Akt inhibitor LY294002³⁶ purchased from Abcam), and D-gal + EVs + DMSO (medium was supplemented with 100 nM D-gal and equivalent volumes of hucMSC-EVs and dimethyl sulphoxide [DMSO]).