P foxo3

The immunoblotting assay was performed according to previous study (Huang et al., 2015 (link)). Briefly, cells were lysed in RIPA buffer with protease inhibitor cocktail (Solarbio, Beijing, China). The bicinchoninic acid method was used to determine the protein content. Proteins were separated by SDS-PAGE gels before electroblotted to PVDF membranes (Millipore, Billerica, MA, United States). The membranes were blocked in 5% fat-free milk for 1 h, followed by incubation with primary antibodies against protein kinase B (AKT; Abcam, Cambridge, UK), p-AKT (Abcam), forkhead box O3 (FOXO3; Abcam), p-FOXO3 (Abcam), Beclin1 (Abcam), microtubule-associated protein light chain 3 (LC3; Cell Signaling Technology, Beverly, MA, United States), and GAPDH (Zhongshan Goldenbridge Biotechnology Co., Beijing, China) at 4°C overnight. The next day, the membranes were washed before incubation with secondary antibodies (Zhongshan Goldenbridge Biotechnology Co.) for 1 h. The blots were detected using an enhanced chemiluminescence substrate kit (Applygen, Beijing, China) and quantified using ImageJ software. The intensity of each signal was normalized to GAPDH values.

GP17 (molecular weight = 947.154; purity > 98%) was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). The positive control drug NBP was obtained from CSPC NBP Pharmaceutical Co., Ltd. Triphenyltetrazolium chloride (TTC) was purchased from Sigma–Aldrich (MO, United States). Primary antibodies against p62/SQSTM1, LC3-B, BNIP3, NAMPT, SIRT1, SIRT2, MnSOD, PGC-1α, FOXO3 and p-FOXO3 were obtained from Abcam (Cambridge, UK). Primary antibodies against Hif1α and Beclin1 were obtained from Proteintech (Wuhan, China). A primary antibody against SIRT3 was obtained from Cell Signaling Technology (MA, USA). The inducer RAP and the inhibitors 3-MA and 2-ME were obtained from MedChemExpress (New Jersey, USA). The inhibitor AGK-7 was obtained from Abcam (Cambridge, UK). ELISA kits for IL-6, TNF–α, MCP-1, T-AOC and 4-HNE were acquired from HaiTai TongDa Sci Tech, Ltd. (Beijing, China).