Fisetin

C2C12 myoblasts (CRL-1772, ATCC, USA) were cultured as previously described [16 (link)]. To analyze the protective effect of fisetin (Sigma-Aldrich Co., USA) on oxidative stress caused by H₂O₂ (Sigma-Aldrich Co.), C2C12 cells were treated with fisetin and H₂O₂ for 24 h, or cells pretreated with fisetin, N-acetyl-L-cysteine (NAC, Thermo Fisher Scientific, USA) or zinc protoporphyrin IX (ZnPP, Sigma-Aldrich Co.) for 1 h were further treated with H₂O₂ for 24 h. To evaluate the inhibitory action of fisetin on ROS accumulation by H₂O₂, cells were treated with fisetin or NAC for 1 h and then treated with H₂O₂ for 1 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Thermo Fisher Scientific) assay was used to investigate the protective potential of fisetin on the suppression of cell viability by H₂O₂ according to the previously described method [17 (link)]. Morphological changes of cells were observed using a phase-contrast microscope (Olympus, Japan). Stock solutions of fisetin and H₂O₂ were prepared by dissolving in dimethyl sulfoxide (DMSO, Sigma-Aldrich Co.), and the highest concentration of DMSO was less than 0.05%, showing no cytotoxicity.

Fisetin was purchased from Sigma–Aldrich (≥98% purity by HPLC; Sigma-Aldrich, St. Louis, MO, USA). Cell viability was detected using the cell counting kit-8 assay kit (CCK-8, Sigma) as described previously [18 (link)]. Briefly, BEAS-2B cells were seeded in a 96-well culture plate at a density of 1 × 10³ cells/well. The cells were treated with 0–100 μM Fisetin for 24 h. Next, CCK-8 detection solution was added to the culture plate and incubated at 37 °C for 4 h. Cell viability was detected using a multimode reader (Thermo, Waltham, MA, USA) based on the absorbance at 450 nm.