Zinc protoporphyrin 9

Penehyclidine hydrochloride injection was purchased from Chengdu List Pharmaceutical Company (Lot No. 121107, Sichuan, China) and was freshly diluted to 0.05 mg/ml in 0.9% NaCl solution at the time of use. Zinc Protoporphyrin IX (ZnPP, a HO-1 inhibitor) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in 0.2 M NaOH, and subsequently diluted to 2.5 mg/ml in 0.9% NaCl solution (pH 7.4) before use. Unless otherwise indicated, all other reagents used in this study were purchased from Sigma-Aldrich.

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.

LiCl (GSK3β inhibitor), MG132 (Proteasome inhibitor), PD98059 (ERK1/2 inhibitor), SB230580 (p38 inhibitor), leptomycin B (LMB, Nuclear export inhibitor), zinc protoporphyrin IX (ZnPP, HO-1 inhibitor), 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-Fluorouracil (5-FU) and oxaliplatin were purchased in Sigma Aldrich (St. Louis, MO, USA). Antibodies against cyclin D1, phospho-cyclin D1 (Thr286), HA-tag, p-GSK3β, total-GSK3β, p-p38, total-p38, HO-1, Nrf2, cleaved PARP, TBP and β-actin were purchased in Cell Signaling (Bervely, MA, USA).