Nrf2 small interfering rna sirna

C2C12 myoblasts were obtained from the Stem Cell Bank, Chinese Academy of Sciences, China (Shanghai, China). High purity hydrogen peroxide (H₂O₂) (30%, 10011218, China) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). TMAO of 95% purity was purchased from Sigma-Aldrich (St. Louis, MO, USA) (317594-5G). The GSH-Px assay kit (A005-1-2) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Nrf2 small interfering RNA (siRNA) was provided by Santa Cruz Biotechnology, Inc. (sc-37049, Dallas, TX, USA). Lipofectamine 2000 (lip2000) for transfection was purchased from Thermo Fisher Scientific (68019, Waltham, MA, USA). Antibodies used in this study include Nrf2 (#12721, CST, USA), HO-1 (#43966, CST, USA), NQO1 (#62262, CST, USA), CAT (21260-1-AP, Proteintech, Wuhan, China), and GAPDH (10494-1-AP, Proteintech, China).

Nrf2 activity was quantified using an antioxidant response element (ARE) reporter HepG2 cell line (BPS Bioscience, San Diego, CA, USA). Cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum. Luciferase activity was measured using the One-Step Luciferase Assay System (BPS Bioscience), and fluorescence intensity was measured using FluoStar Optima (BMG Labtech, Ortenberg, Germany). Cells were transfected with Nrf2 small interfering RNA (siRNA) (Santa Cruz Biotechnology, Dallas, TX, USA) or control siRNA for 24 h by using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Tolfenamic acid (purity ≥98%) was purchased from a commercial supplier (Abcam, Cambridge, MA, USA), and 3-nitropropionic acid (3-NP) was purchased from Sigma-Aldrich (MO, USA). The mouse monoclonal antibody against huntingtin (EM48) was purchased from Millipore (CA, USA). The rabbit polyclonal antibodies against LC3, P62, and HO1; the mouse monoclonal antibody against β-actin; and horseradish peroxidase-conjugated secondary antibodies were purchased from Proteintech (Wuhan, China). ML385, a specific Nrf2 inhibitor, was purchased from MedChem Express (NJ, USA). Nrf2 small interfering RNA (siRNA) and rabbit polyclonal antibody against Sp1 were purchased from Santa Cruz Biotechnology Inc. (CA, USA). The rabbit polyclonal antibody against NQO1 and D,L-buthionine-(S,R)-sulfoximine (BSO), a specific glutathione synthase inhibitor, was purchased from Abcam (Cambridge, MA, USA).