2.1. Cell Culture and Induction of Oxidative Stress

Human HEK293T cells were cultured in low glucose Dulbecco’s Modified Eagle Medium (DMEM, BioWest, Nuaille, France; L0103-500) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were incubated at 37 °C and 100% humidity with 8.5% CO₂. To induce oxidative stress, cells were treated at 80% confluency with menadione, H₂O₂, or KBrO₃ using the concentrations and times given in the results. In experiments where a recovery phase was included, DMEM with the oxidizing agent was gently removed and replaced with fresh DMEM for up to 48 h. As the cells would have grown confluent in the retreatment experiments, they were split 1:3 in fresh medium after the first treatment.

For growth curve experiments, 10⁵ cells were seeded into 6-well plates and grown for 24 h before addition of the oxidizing agents. At 0 and 24 h, cells were detached with trypsin/EDTA and counted using a Luna-FL cell counter (Logos Biosystems, Gyeonggi-do, Korea) with four biological replicates.