Phospho nrf2

Luteolin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, 2′,7′-dichlorofluorescein diacetate (DCF-DA), a mouse/rabbit red starter duolink kit, and propidium iodide (PI) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Luteolin was prepared in 100% dimethyl sulfoxide to obtain a 100 mg/ml stock solution, which was aliquoted and stored at −20 °C for 3–6 months for use in the various experiments. Primary antibodies against p53, p21, Bcl-2, Bax, caspase-9, glutamate cysteine ligase catalytic (GCLc), glutathione synthetase (GSS), catalase, heme oxygenase-1 (HO-1), Nrf2, TET1, TET2, TET3, DNMT3B, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DNMT1, phospho-Nrf2, and TATA box-binding protein (TBP) were purchased from Abcam (Cambridge, MA, USA), and caspase-3 and DNMT3A were purchased from Cell Signaling Technology (Beverly, MA, USA).

The total protein content of the cells was isolated using RIPA lysis buffer (Beyotime) with 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime), and protein concentration was measured using a BCA protein assay kit (Beyotime). For each protein sample, 30 µg was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime), followed by transfer to a polyvinylidene difluoride membrane (PVDF; Bio-Rad). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against 3-NT (1:2000) (Abcam), 4-Hydroxynonenal (4-HNE) (1:500) (Abcam), Phospho-Nrf2 (1:5000) (Abcam), Nrf2 (1:1500) (Abcam), HO-1 (1:2000) (Abcam), Phospho-Akt (1:1000) (Cell Signaling Technology, MA, USA), Akt (1:1500) (Cell Signaling Technology), and GAPDH (1:1000) (ZenBio, Chengdu, China) overnight at 4 °C, followed by the respective secondary antibodies: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:3000) (ZenBio) or HRP-conjugated goat anti-mouse IgG (1:3000) (ZenBio). Bands were detected using electrochemiluminescence plus reagent (Bio-Rad, USA). Finally, the intensity of these bands was quantified using ImageJ software (Version 1.8.0; National Institutes of Health, USA).