Nrf2 antibody

The cells were first processed using enzymatic chromatin IP kit (Cell signaling technology), according to the manufacturer’s instructions. Nrf2 antibody (Cell signaling Technology) was used for ChIP analysis. The ChIP procedure was performed by PCR using the following human GSS promoter-specific primers: forward 5′-CTGGGAATAACCAGACACCTA-3′ and reverse 5′-CAGGTTCAAGCAATTCTCCTG-3′. The PCR cycle conditions were as follows: initial denaturation at 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 7 min. The amplified products were resolved using electrophoresis and analyzed using image analysis software.

Cells (5 × 10⁵ per well) were seeded into six well plates containing a coverslip. The next day, cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by permeabilization with 0.1% sodium citrate with 0.1% Triton X-100. The cells were incubated with Nrf2 antibody at 1:1000 dilution (12721S; Cell Signaling, Danvers, MA, USA) for 1 h at room temperature. The cells were then washed with cold PBS three times (5 min each) and incubated with Alexa 514-labeled anti-rabbit secondary antibody (1:1000) (A11061; Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. The cells were then incubated with conjugated actin antibodies for 15 min. After washing with PBS, the coverslips were mounted onto the slides with ProLong Diamond Antifade Mountant with DAPI (P36962; Thermo Fisher Scientific, Waltham, MA, USA). Standard immunofluorescence methods were used as described previously [28 (link)]. Imaging was performed using EvosFL fluorescence microscope (Life Technologies, Carlsbad, CA, USA) at 40× objective magnification. Fluorescence intensities from images of six randomly selected microscopic fields of cells were semi-quantitatively analyzed by densitometry (ImageJ software, NIH).

C₃H₁₀T_1/2 cells were plated in a 48-well plate and cultured with 0, 10^− 8, 10^− 7, or 10^− 6M Tanshinone IIA for 24 h, followed by culture in fresh medium supplemented with 500µM H₂O₂ for another 24 h. After fixation with 4% formaldehyde solution at 37℃ for 15 min, the cells were incubated with an Nrf2 antibody (12721, Cell Signaling Technology, Danvers, Massachusetts, USA, 1:500) overnight at 4℃. Cells were further incubated in CoraLite594-conjugated goat anti-rabbit IgG (SA00013-4; Proteintech, Wuhan, Hubei, China; 1:200) for 1 h at 37℃ in the dark. Antifade mounting medium with 4,6-diamidino-2-phenylindole (DAPI) was used for nuclear fluorescence staining (H-1200, Vector Laboratories, San Francisco, California, USA).