Mnsod

Sweroside (purity > 98%, Figure 2A) was obtained from National Institutes for Food and Drug Control (Beijing, China). Fetal bovine serum (FBS) and phosphate buffer saline (PBS) were acquired from Servicebio Company (Wuhan, China). DMEM, DMSO, trypsin, the RIPA Lysis buffer and the PVDF membrane were obtained from Solarbio Company (Beijing, China). The annexin V-FITC apoptosis detection kit was obtained from Vazyme Biotech Company (Nanjing, China). Hoechst 33342, NAM, SIRT1, NF-κB, β-actin and the proliferating cell nuclear antigen (PCNA) antibodies were from Beyotime Institute of Biotechnology (Shanghai, China). The TNF-α, IL-1β, IL-6, IL-10, MnSOD, FOXO1, COX-2 and i-NOS antibodies were obtained from Proteintech Company (Wuhan, China). The ROS assay kit, CCK-8 assay kit, NO assay kit, cell cycle and apoptosis analysis kits were from Beyotime Institute of Biotechnology (Shanghai, China). The PGE2 Elisa Kit was purchased from Hui Jia Company (Xiamen, China). The other chemicals were of analytical grade and were commercially available.

Western blotting was performed as previously described (Wang et al., 2015 (link)). Briefly, cell protein extracts (15–20 μg) were separated by 10–15% SDS-PAGE and transferred to PVDF membranes. Proteins were detected with the following antibodies: SIRT3 (1:1000, Cell Signaling Technology, Boston, MA, USA), MnSOD (1:1000, Proteintech, Chicago, IL, USA), cyclophilin D (CypD; 1:1000, Abcam, Cambridge, UK), cytochrome C (Cyt C; 1:1000, Cell Signaling Technology), cleaved caspase-3 (1:1000, Cell Signaling Technology), Bax (1:1000, Cell Signaling Technology, Boston, MA, USA), B-cell lymphoma 2 (Bcl-2; 1:1000, Cell Signaling Technology, Boston, MA, USA), COX-IV (1:1000, Abcam, Cambridge, MA, USA) and GAPDH (1:2000, Proteintech, Chicago, IL, USA).

Western blotting was conducted with primary antibodies against p66Shc (BD Biosciences, USA), MnSOD (Proteintech Group, Wuhan, China), catalase (Abcam Ltd., Cambridge, UK), NOX2 (Abcam Ltd.), and β-actin (ZSGB-BIO, Beijing, China). Protein quantification was performed using Gel-Pro Analyzer version 4.0 (Media Cybernetics, MD, USA).