Sa β gal staining kit

To evaluate cellular senescence, β-gal activity was analyzed using a SA-β-Gal staining kit (Biyuntian, Beijing, China), following the manufacturer’s instructions. Briefly, pMSCs at P5, P10, and P15 were plated in a 6-well plate, fixed with fixative solution for 15 min at room temperature, and washed three times with PBS. The cells were incubated overnight with freshly prepared staining solution at 37 °C in the absence of CO₂. After washing with 70% ethanol, the aging cells were dyed blue. The number of these blue cells was counted under a inverted phase-contrast microscope (Nikon, Tokyo, Japan).

High-sugar DMEM medium (PYG0073), 0.25% trypsin solution (AR1007) and CCK-8 kit (AR1199) were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). Fetal bovine serum (FBS) (SA201.01) was obtained from CellMax Technology (Beijing) Co. (Beijing, China). Penicillin mixture (100×) (P1400) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The PEE was generously donated by Prof. Li-Ming Wu of the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, and was analyzed via high performance liquid chromatography-diode array detection/quadrupole time-of-flight mass spectrometry (HPLC-DAD/Q-TOF-MS) to determine the PEE components (Table S1) [43 (link)]. The D-gal (478518-63-7) was obtained from Sigma-Aldrich (St Louis, MI, USA). SA-β-Gal staining kit (C0602) was acquired from Beyotime Biotechnology (Shanghai, China). Malondialdehyde (MDA) (A003-3-1), peroxidase (CAT) (A007-1-1), glutathione peroxidase (GSH-Px) (A005-1-2), superoxide dismutase (SOD) (A001-1-2), total antioxidant capacity (T-AOC) (A015-2-1), reactive oxygen species (ROS) (E004-1-1) biochemical kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Nrf2-specific inhibitor ML385 (HY-100523) was purchased from MedChemExpress LLC (Shanghai, China). Information on the antibodies used in the experiment is shown in Table 1.

RNA extraction kit was from Qiagen (Qiagen Inc., CA, USA). DAPI (#4083) and antibodies against Nrf2 (#12721), Akt (#9272), HO-1 (#43966), CAT (#14097), GSK-3β (#12456), NQO1 (#62262), P16 (#80772), P21 (#2947), p-Akt (#4060), p-GSK-3β (#5558), Fyn (#4023), Histon H3 (#4499), and β-actin (#4970) were obtained from Cell Signalling Technology Corporation (MA, USA). CD4⁺ T Cell Isolation Kit II was obtained from Miltenyi (Bergisch Gladbach, Germany). SA-β-Gal staining kit was obtained from Beyotime (Shanghai, China). LY294002 (L9908) and ML385 (SML1833) were obtained from Sigma-Aldrich (St. Louis., MO, USA).

NP cell senescence was analyzed using a senescence associated β-galactosidase (SA-β-Gal) staining kit (Beyotime, China). Briefly, NP cells were washed with PBS for three times and fixed with fixative for 15 min, followed by rinsing with PBS for three times (3 min for each time). Thereafter, NP cells were incubated with staining working solution overnight at 37°C under seal condition. Finally, SA-β-Gal activity expressed as the staining-positive NP cells to the total NP cells was analyzed using the Image-Pro Plus software (Version 5.1, Media Cybernetics, Inc.).

The cells were subjected to the specified treatments, and the SA-β-gal activity was assessed using the SA-β-gal Staining Kit (Beyotime, Nanjing, China), following the manufacturer's instructions. The levels of ROS were determined using the ROS assay kit (Solarbio, Beijing, China), following the manufacturer's instructions. Red fluorescence emitted as a result of dihydroethidium bromide oxidation was employed to quantify the level of superoxide radical (O2•-) level in the cells. For each sample, 1 × 10⁴ cells were harvested from the gated cells and fluorescence was immediately measured using flow cytometer (BriCyte E6, Shenzhen, China).