D8310

The senescence of C6 cells was induced by D-gal (D8310, Solarbio) treatment at 222 mM for 8 days, or by etoposide (E1383, Sigma) at 6 μM for 1 day, then recovered after a further 4 days. The senescence of U87MG cells was induced by D-gal treatment at 222 mM for 7 days. To rescue D-gal-induced glioma cell senescence, metformin (HY-17471A, MedChemExpress) was dissolved in water (1 M in stock solution) and applied at the concentration of 2 mM for 1 day.

EBs with uniform size were randomly divided into three groups including the Control, the D-gal and RSV group. RSV group included RSV-L (2 μm/L), RSV-M (10 μm/L) and RSV-H (50 μm/L). The following is the detail of grouping: (1) control group: the EBs were cultured with regular medium. (2) D-gal group or nervous injury group: the EBs were exposed to 5 mg/ml D-gal (Solarbio, D8310) in medium from day 3, in order to induce nervous injury. (3) RSV groups: after D-gal for 24 hrs, the cultures with the continuation of D-gal were exposed to RSV (Sigma-Aldrich, R5010) in medium with three different doses, such as 2 μm/L, 10 μm/L and 50 μm/L. The cultures with uniform size were harvested at D5, D8, D12 and D40 for follow-up experiments. The number of specimens for each group is greater than or equal to 5 at each age (N ≥ 5) (only 5 specimen could be chosen in RSV-H at D40).