2.6. NRF2 nuclear translocation by immunofluorescence and western blotting

For immunofluorescence assays, HCT116 cells were seeded in glass-bottom petri dishes at a concentration of 2 × 10⁴ per well with DMEM containing 10% FBS and 1% antibiotics (equal mixture of 0.5% penicillin and 0.5% streptomycin) overnight. The cells were then incubated with or without 10 μM RUT for 6 h and fixed with 4% paraformalde-hyde for 10 min. The cells were blocked with 3% bovine serum albumin in TBS (Tris buffered saline) for 40 min, and then incubated with NRF2 antibody (dilution of 1:100) overnight at 4 °C. After washing three times with TBS containing 0.1% Tween 20, the cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated goat-anti-rabbit antibody for 2 h and washed 3 times with TBS containing 0.1% Tween 20. Finally, the cells were stained with 10 μg/mL Hoechst33342 for 15 min and examined with a 3D living cell high speed multicolor laser scanning confocal microscope (Perkin Elmer Life Science, Cambridge, UK) after washed thrice with phosphate-buffered saline.

For western blots, HCT116 cells were seeded into 6-well dishes at a concentration of 2 × 10⁵ per well with DMEM containing 10% FBS and 1% antibiotics. After attachment, cells were incubated with SFN (10 μM) or RUT (5 μM and 10 μM) for 24 h. The Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) was used to isolate the nuclear and cytosol protein according to the manufacturers protocol. The concentrations of collected protein were determined with the bicinchoninic acid assay and the samples were stored at −80°C until use. ProteinSimple Wes protein analysis (San Jose, CA) was used to detect NRF2 levels according to the user’s guide.