N6022

Cell culture reagents were purchased from GE and fetal bovine serum (FBS) was purchased from PAN-Biotech; DTT was purchased from Millipore (Massachusetts, USA); Compound C, AICAR, Resveratrol, Suramin, SNAP, and ODQ were purchased from Sigma-Aldrich; N6022, Pifithrin-α, VO-Ohpic trihydrate, AS1842856, and MK 2206 were from MCE. Hydrogen peroxide (H₂O₂) was from Sinopharm Chemical Reagent; 3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sangon Biotech; Lipofectamine® 3000 and Attractene were from Thermo Fisher Scientific and Qiagen, respectively. Fluor-de-Lys SIRT1 fluorometric drug discovery assay kit was from Enzo Life Sciences; Hydrogen Peroxide Assay Kit, Nitric Oxide Assay Kit, RIPA lysis buffer, Bicinchoninic Acid assay Kit, and Nuclear and Cytoplasmic Protein Extraction Kit were from Beyotime; Super ECL Prime was from US Everbright®Inc. The mRNA extraction kit was from Bioteke. Protein A Magnetic Beads and the Muse Count & Viability Assay Kit were purchased from Millipore. Anti-Flag M2 Magnetic Beads was from Sigma-Aldrich. Antibodies for phospho- and total-AMPK, acetyl- and total-p53, SIRT1, Cleaved- and total-Caspase-3, FOXO1, PTEN, β-actin, and acetylated-Lysine were from Cell Signaling Technology; antibodies for P21 was from Abcam; antibodies for Prdx2 was from Santa Cruz Biotechnology.

H₂O₂, KU55933, AZD7762, N6022, PMA, ionomycin, pifithrin‐α, CCCP, cycloheximide, trigonelline, dipropylenetriamine (DPTA) NONOate, hydroxyurea, neocarzinostatin, trypan blue, antimycin A, oligomycin A, and salts used for buffers were from Sigma‐Aldrich. All the compounds were used in a range of concentrations that did not per se induce any significant modulations of GSNOR levels (Appendix Fig S6), i.e., KU55933, 5 μM; AZD7762, 20 nM; and pifithrin‐α, 20 μM. Other concentration used are as follows: H₂O₂, 100, 200 or 400 μM; cycloheximide, 30 μM; trigonelline, 2.5 μM; neocarzinostatin, 0.5 μg/ml; DPTA, 400 μM; HU, 2 mM; CCCP, 5 or 10 μM; N6022, 25 μM; PMA, 200 ng/ml; ionomycin, 300 ng/ml; antimycin A, 1 μM; and oligomycin A, 1 μM, unless otherwise indicated.