Bicinchoninic acid quantitation kit

Cells and ovarian tissues were lysed in radioimmunoprecipitation assay buffer with protease inhibitor cocktail and PhosSTOP (Roche, Basel, Switzerland). Protein extraction was analyzed using a bicinchoninic acid quantitation kit (Beyotime, Beijing, China). After 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane with primary antibodies, followed by horseradish peroxidase–linked secondary antibodies (Abcam, Hongkong, Chian) and enhanced chemiluminescence reagents (Millipore Corp., Billerica, MA, USA). All antibodies used are listed in Supplementary Table 2.

As described in the related literature, EVs were isolated from the supernatant of rat UC MSCs (Shao et al., 2018). After the UC-MSCs reached about 80% confluency, the culture medium was carefully collected and centrifuged at 300 × g for 10 minutes, followed by a 2000 × g centrifugation for 15 minutes (4°C). The cell supernatant was then collected, and the cellular debris was discarded. The supernatant was then centrifuged at 10,000 × g for 45 minutes, followed by a centrifuge at 120,000 × g for 70 minutes at 4°C with an ultracentrifuge. The precipitation was collected and resuspended with 3 mL phosphate-buffered saline (PBS), and centrifuged again at 120,000 × g for 70 minutes (4°C). After supernatant removal, the precipitation was resuspended with 200 μL PBS. The protein content of the EV suspension was calibrated using a bicinchoninic acid quantitation kit (Beyotime, Shanghai, China). The expression of CD81, a specific marker for EV (Kowal et al., 2016; Kowal et al., 2017), was tested using a Western blot assay. The culture medium deprived from EVs was also assessed for CD81 expression as the negative control.