Trans 1 2 methoxyvinyl pyrene

Pba was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NRF2 antibody was obtained from Abcam (Cambridge, MA, USA). Antibodies recognizing AKR1C1 and BCRP were purchased from Abnova (Taipei City, Taiwan) and Cell Signaling Technology (Beverly, MA, USA), respectively. PRDX3 and β-tubulin antibodies were obtained from Santa Cruz Biotechnology. Ko143, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and puromycin were obtained from Sigma-Aldrich (Saint Louis, MO, USA). 5(6)-Carboxy-2′,7′-dichlorofluorescein diacetate (DCFDA), trans-1-(2′methoxyvinyl) pyrene, and MitoGreen were purchased from Life Technologies (Carlsbad, CA, USA). The lentiviral system containing a pre-designed human NRF2 shRNA and nonspecific scrambled RNA (scRNA) was bought from Sigma-Aldrich.

The cells were seeded in a cover glass dish (SPL Life science, Gyeonggi-do, South Korea) and cultured for overnight. After the Pba-laser irradiation, the cells were washed with PBS and incubated with 50 µM trans-1-(2′methoxyvinyl) pyrene (Life Technologies) for 30 min. For the nuclei staining, Hoechst 33342 (H342) was added to the above dishes and incubated for 10 min. Green fluorescence from singlet oxygen was detected immediately using a LSM 710 confocal microscope (Carl Zeiss, Jena, Germany) and intensities were quantified using a ZEN2011 software (Carl Zeiss).