Srt1720

HEK 293T cells were plated in 24-well plates 24 h before transfection. When the cells reached 40–50% confluence, they were transfected with a plasmid containing the promoter sequence of NF-κB using Lipofectamine 3000 transfection reagent (Invitrogen). Ex527 (40 μM; Selleckchem) and SRT1720 (2 μM; Selleckchem) were added at the same time. Twenty-four hours later, the cells were lysed in 1 × PLB and luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) as recommended by the manufacturer. All luciferase assay experiments were performed in triplicate.

Cells were treated with 1 mM H₂O₂ (Sigma-Aldrich, USA) for 2 h. To activate mitochondrial biogenesis, cells were preincubated with 5 μM resveratrol (Sigma-Aldrich) or 0.5 μM SRT1720 (Selleck, USA) for 24 h. Cell viability was detected using CCK-8 kits (DOJINDO, Japan) according to the manufacturer's protocols. Briefly, cells were plated into a 96-well plate. At the indicated time after treatments, 10 μl CCK-8 solution was added into the wells. Cells were then incubated at 37°C in an incubator for 1 h. Then, a microplate reader (Labsystems Dragon, Finland) was used to measure the absorbance at 450 nm.

The HEK293T cell line was a gift from the Zhao lab of Fudan University (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen), penicillin (Invitrogen) (100 U/ml), and streptomycin (Invitrogen) (100 U/ml). Full-length PEPCK1 (wildtype, 3K/R and 3K/Q) and SIRT2 (wildtype and H187Y) plasmids were also gifts from the Zhao lab of Fudan University (Shanghai, China). Plasmids were cloned to Flag- or HA-tagged destination vectors according to different needs. Point mutations for PEPCK1 and SIRT2 were generated by site-directed mutagenesis. Antibodies against Flag (Sigma, St. Louis., MO, USA), HA (Santa Cruz, Dallas, TX, USA), PEPCK1 (Santa Cruz), SIRT2 (Sigma), α-Tubulin (CST, Danvers, MA, USA), acetylated α-Tubulin (Abcam, Cambridge, UK), and β-actin (Sigma) were all purchased, and the polyclonal antibody against acetyl-lysine was a gift from the Zhao lab. Trichostatin A (CST), nicotinamide (Sigma), sirtinol (Selleck, Houston, TX, USA), MG132 (Sigma), EX527 (Selleck), SRT1720 (Selleck), and CHX (Sigma) were all purchased. Control and siSIRT2 adenovirus were purchased (Vector Biolabs, Malvern, PA, USA).

HEPES sodium salt, HEPES, potassium chloride, calcium chloride dihydrate, D-(+)-glucose, collagenase V, insulin, rosiglitazone, ZnCl₂, ZnSO₄ and TPEN (an intracellular membrane-permeable chelator for various ions, including zinc) were purchased from Sigma (Saint Louis, MO). Total OXPHOS Rodent WB was purchased from Abcam (Cambridge, MA). The Dual Luciferase Assay Kit was obtained from Promega (Madison, WI). Fluo-4 AM, cell-permeant Rhodamine 123 and 2-NBDG were purchased from ThermoFisher Scientific (Waltham, MA). SRT1720 and ZLN005 were purchased from Selleck (Houston, TX). The zinc detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, JS, China).