Sirt3

The expression of cellular SIRT1, SIRT3, and NMNAT1 was determined by western blot assay. Rat RCECs were grown at a density of 1 × 10⁶ cells/cm² in T-25 cm² flasks and were then exposed to various experimental conditions. For the western blot assay, cells were prepared in lysis buffer (50 mM Tris [pH=7.5], 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EDTA, and 0.1% SDS), and the protein concentrations were determined with a BCA protein assay. Equal amounts of the whole protein extract (8 μg) underwent electrophoresis on a 10% SDS-polyacrylamide gel and were transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore™, Billerica, MA, USA). After transfer, the membranes were incubated overnight at 4 °C with antibodies against NMNAT1 (Bioss Inc., Beijing, China), SIRT1 (Affinity Biosciences, Cincinnati, OH, USA), SIRT3 (ProteinTech Group, Chicago, IL, USA), and GAPDH (Cell Signaling Technology, Danvers, MA, USA). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence western blotting detection reagents. The relative densities of the bands were determined by image analysis with Image-Pro Plus 6.0 software.

Total proteins were extracted using ice-cold RIPA buffer containing protease inhibitor cocktail (Roche). Protein extraction kit (Beyotime) was used to extract nuclear and cytoplasmic protein. The concentrations were determined using a BCA kit (Beyotime). Equal amount of protein from each sample was run in 8–12% Tris-glycine SDS-PAGE gel, followed by transfer to PVDF membrane (Millipore). Membranes were blocked with 5% skim milk and probed with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-IgG (Beyotime) for 1 h at room temperature. The intensity of bands was visualized and determined using an enhanced chemiluminescence detection system (Bio-Rad Laboratories). Primary antibodies used were: γ-H2AX, GPX-1, Akt, p-Akt, p21, S6, p-S6, p-LKB1, LC3B, AMPK, p-AMPK, Atg7, p62, ACC, p-ACC (1:1000, Cell Signaling), SOD1, SOD2, Catalase, p-NRF2, FOXO3a, p-FOXO3a (1:1000, Abcam), p16, NRF2, Lamin B1, GCLC, GCLM, HMOX1, NQO1, TXN, Keap1, SIRT1, SIRT3, SIRT6, LKB1, CAMKK2(1:1000, ProteinTech), Actin and GAPDH (1:1000, Beyotime). The primary images (Supplementary Fig. 18) were cropped for presentation.