E1z6r

The chemicals and reagents used in this study were (1) RTA dh404, which was purchased from Cayman Chemicals; (2) PrestoBlue™ Cell Viability Reagent from ThermoFisher/Invitrogen; (3) fetal bovine serum (FBS), the antibiotics penicillin/streptomycin (P/S), and modified Eagle medium (MEM) were purchased from Gibco and Roswell Park Memorial Institute (RPMI) 1640 medium (USA); (4) phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), trypsin-EDTA (0.25%), and Trypan Blue Solution were purchased from Sigma (St Louis, MO); (5) the polyvinylidene fluoride membrane (PVDF) (Millipore) and molecular weight markers were purchased from Bio Rad (USA); and (6) propidium iodide (PI) (USA).
Western blot antibodies were also purchased from commercial vendors and used at the indicated dilutions: Cyclin B (1:1000; Proteintech; 55004-1-AP), CDK1 (1:1000; Cell Signaling; E1Z6R), Wee1 (1:1000; Proteintech 14375-1-AP), p21 (1:1000; Cell Signaling E2R7A); Bcl-2 (1:1000), Nrf2 (1:1000; Cell Signaling; D1Z9C), Bcl-2(1;1000; Proteintech), Bax (1:1000; Proteintech), Caspase-3 (1:1000; Affinity), PARP (1:1000; Affinity), LC3B (1:1000; Invitrogen), p62/SQSTM1 (1:1000; Affinity), and β-actin (1:20000; Sigma; A5441).

Western blotting was performed as previously described [38 (link),39 (link)]. All cell lysates (50–80 µg) were prepared in an ice-cold lysis buffer. Protein samples were loaded onto 10–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis membranes for electrophoretic separation and then transferred to PVDF membranes (Millipore) at 500 mA for 2 h. After blocking the buffer overnight with 5% non-fat dry milk in PBS containing Tween20 (PBST), the membranes were incubated with primary antibodies [Cyclin B1 (1:1000; Proteintech; 55004-1-AP), CDK1 (1:1000; Cell Signaling; E1Z6R), NRF2 (1:1000; Proteintech; 16396-1-AP), and β-actin (1:20,000; Sigma; A5441)] for 2 h at room temperature or overnight at 4 °C. Membranes were then washed once with PBST and twice with PBS, incubated with the secondary antibody Li-COR (Taipei, Taiwan) at a 1:20,000 dilution for 30–40 min, and washed again. Antigens were visualized using a near-infrared fluorescence imaging system (Odyssey LICOR, USA), and these data were interpreted using Odyssey2.1 software or a chemiluminescence detection kit (ECL; Amersham Corp., Arlington Heights, IL, USA). Densitometry analysis (including the integrated density of bands) was carried out using ImageJ (NIH), followed by normalization of the measured values to β-actin as a loading control.

All samples were lysed in 200 μl of lysis buffer. A total of 50–75 μg of protein per sample were loaded onto 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis membranes for electrophoretic separation and then transferred to PVDF membranes and subjected to electrophoresis at 50 V for 4 h. After blocking overnight with Odyssey blocking buffer (USA), the membranes were incubated with primary antibodies [Cyclin A2 (1:1,000; proteintech; 18202-1-AP), Cyclin B1 (1:1,000; proteintech; 55004-1-AP), CDK1 (1:1,000; cell signaling; E1Z6R), NRF2 (1:1,000; proteintech; 16396-1-AP), CHK2 (1:1,000; abgent.com; AP4999a), p-CHK2 (1:1,000; abgent; AP50241), CHK1 (1:1,000; proteintech; 22018-1-AP), p21 (1:1,000; Cell Signaling; #2947), and β-actin (1:20,000; Sigma; A5441)] for 2 h at room temperature. Subsequently, the membranes were washed several times and then incubated with a corresponding secondary antibody (IRDye Li-COR, USA) at a dilution of1:20,000 for 30–45 min. Antigens were then visualized using a near-infrared fluorescence imaging system (Odyssey LICOR,USA), and the data were interpreted using the Odyssey2.1 software or a chemiluminescence detection kit (ECL; Amersham Corp., Arlington Heights, IL, USA).