D121053

Total cell extracts were prepared in 1× sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Cell fractions were extracted with nuclear and cytoplasm protein extraction kit (Wanleibio, China). Cell proteins were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with GAPDH (1:1000, BM3876, Bosterbio, USA), p-PERK, p-IRE1, ATF4, P16 (1:500, bs-3330R, bs-16698R, bs-1531R, bs-20656R, Bioss, China), H3, NRF2 (1:500, D153567, D121053, Sangon Biotech, China), and TNF-α, IL-6, P21, MT1, MT2, HRD1, VCP, P65, p-P65, IKK (1:200, sc-52746, sc-32296, sc-136020, sc-13180, sc-13177, sc-293484, sc-136273, sc-514451, sc-166748, sc-7606, Santa Cruz, USA). Secondary anti-rabbit, anti-mouse antibodies (1:1000, BM2004, BA1001, Bosterbio, USA) conjugated with horseradish peroxidase were used. Detection was performed using a Thermo Scientific Pierce enhanced chemiluminescence western blotting substrate (Thermo Scientific). Results were analyzed by Tanon-410 automatic gel imaging system (Shanghai Tianneng Corporation, China).

Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature (RT) for 10 min, washed three times with PBS, and then permeabilized for 15 min with 0.1% Triton-X 100 (Sigma-Aldrich, St. Louis, MO) in PBS at RT. Cells were blocked with PBS supplemented with 4% bovine serum albumin for 30 min and incubated with primary antibodies against NRF2 (1:200, D121053, Sangon Biotech, China), and γH2AX (1:200, sc-517348, Santa Cruz, USA) at 4 °C for 16 h. After washing with PBS three times, cells were incubated with secondary antibodies for 1 h at 37 °C in the dark. Following another three washing steps in PBS, nuclear counterstaining was performed with 1 μg/mL Hoechst 33342 (Sigma Aldrich). Fluorescence images were obtained by Evos f1 fluorescence microscope (AMG, USA).