Sulforaphane

8-week-old male Sprague-Dawley rats were used. Rats were housed by two per cage in a temperature-controlled room. Food pellets and tap water were supplied freely. A total of 18 rats were randomly divided into three groups: (1) sham-operated rats; (2) BOO rats; and (3) BOO rats treated with Sulforaphane (0.5 mg/kg/day) intraperitoneally for 4 weeks. Sulforaphane was provided by Cayman Chemical (USA). Sulforaphane treatment was initiated immediately following the operation of BOO rats. The dose of 0.5 mg/kg/day SFN in this research has been proved effective in other researches. All experimental procedures were approved by the Animal Research Ethics Committee of Shanghai Jiao Tong University School of Medicine.

Male C57BL/6 mice (3–4weeks) were housed in a specific-pathogen-free (SPF) environment at the Laboratory Animal Center of Chinese Academy of Medical Sciences Institute of Radiation Medicine, with a 12 h light/dark cycle and access to food and water freely. The animal chow was purchased from Hua Fu Kang (Beijing, China). After acclimation for 1 week, the mice were randomly classified into two weight-matched groups: (1) normal chow diet (NCD) group (n = 10/group); (2) high-fat diet (HFD) group (n = 20/group) (HFD: protein 18.1%, fat 61.6%, carbohydrates 20.3%). After 22 weeks of the initial feeding period, HFD group mice were randomly assigned to two groups (n = 10/group): a HFD with saline, and a HFD with Sulforaphane (purity >99%, Item NO.10496, Cayman) 25 mg/kg intervention by gavage daily for 6 weeks. The liver tissues were harvested, weighed, and stored at −80°C for further analysis. All animal welfare and experimental procedures complied with the Laboratory Animal Management Regulations in China.

Sulforaphane was provided by Cayman Chemical (USA). Anti-Nrf2, HO-1, and NQO1 antibodies were obtained from Abcam (Cambridge, UK); anti-bcl-2, Bax, PCNA, and GAPDH antibodies were acquired from CST (Danvers, MA, USA); goat-anti-rabbit IgG-HRP, goat-anti-mouse IgG-HRP, and DAB detection Kit were obtained from DAKO. The TdT-mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit was provided by KeyGEN BioTECH (Nanjing, China). The BCA assay kit was provided by Thermo Scientific. The nuclear protein extraction kit was provided by Beyotime Institute of Biotechnology (Beijing, China). The malondialdehyde (MDA) assay kit, total superoxide dismutase (SOD) assay kit, glutathione peroxidase (GSH-Px) assay kit, and catalase (CAT) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Paraquat (PQ)(Sigma Aldrich) was diluted from a 0.1 M stock prepared with ultrapure water (Invitrogen). Antibodies for western blotting and immunocytochemistry: rabbit anti‐catalase (#12980), rabbit anti‐VDAC (#4866) and rabbit anti‐Caspase 3 (#9662) from Cell Signaling; rabbit anti‐catalase (sc‐50508), mouse anti‐p53 (sc‐126) and rabbit anti‐Nrf2 (sc‐722) from Santa Cruz; mouse anti‐ATP5α (ab14748) and rabbit anti‐catalase (ab16731) from Abcam; rabbit anti‐phosphoNrf2ser40 (bs‐2013R) from Bioss; mouse anti‐ PKCδ (cat #36520) from Transduction Laboratories; mouse anti‐Tubulin (ATN01) from Cytoeskeleton. Inhibitors: sodium azide from Fisher Scientific; PKCδ (Gö6983), PKCα/β (Gö6976) and Caspase 3 (Z‐DEVD‐FMK) from Calbiochem. Fluorescent Dyes: propidium iodide (PI), 2′,7′‐dichlorodihydrofluorescein diacetate (DCF), tetramethylrhodamine methyl ester perchlorate (TMRM) from Molecular Probes. Mitochondrial targeted antioxidants: MitoQ, 1 mM stock in H₂O; and MitoTEMPO, 2 mM stock in H₂O, from Sigma‐Aldrich. Nrf2 activators: Oltipraz (Tocris) 5 mM stock in DMSO; Sulforaphane (Cayman Chemical), 5 mM stock in ethanol.

The growth inhibition assay was performed as described previously [21 (link)]. Briefly, 2 × 10⁴ cells were seeded into 24-well plates and cultured for 96 or 192 h in a growth medium containing various concentrations of bleomycin (Wako Pure Chemical), etoposide (BioVision, Mountain View, CA, USA), cisplatin (Sigma-Aldrich), camptothecin (Sigma-Aldrich), sulforaphane (Cayman Chemical Company, Ann Arbor, MI, USA), resveratrol (Wako Pure Chemical), quercetin (Sigma-Aldrich), kaempferol (Sigma-Aldrich), or genistein (Wako Pure Chemical). Subsequently, cell growth was measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Fitchburg, WI, USA).
The colony formation assay was performed as described previously [21 (link)]. Briefly, 1 × 10²–10⁵ cells were seeded into 60 cm dishes containing 5 mL of agarose medium with various concentrations of methyl methanesulfonate (MMS; Sigma-Aldrich). After incubation for 2 weeks at 37 °C, visible colonies were counted, and the survival percentage was calculated by comparing the number of surviving colonies with that corresponding to the untreated controls.