Fig. 3: Nuclear factor erythroid 2-related factor 2 (Nrf2) scavenges reactive oxygen species (ROS) via glutathione (GSH) depending on glycolytic metabolism in radioresistant oral squamous cell carcinoma cells (OSCC).

A, D The glycolysis stress test was performed using a Seahorse XF24 analyzer. A The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using HSC-2-WT cells and HSC-2-R cells before and after treatment with glucose and 2-deoxyglucose (2-DG). At 48 h after transfection, HSC-2-WT and HSC-2-R cells were seeded, after which the glycolysis stress test was performed. D The glycolysis stress test was performed using SAS-WT cells and SAS-R cells in the same way as in A. B, C, E, F The mRNA levels of malic enzyme 1 (ME1) and glutamate-cysteine ligase catalytic subunit (GCLC)/glutamate-cysteine ligase modifier subunit (GCLM) were investigated using real-time quantitative polymerase chain reaction (RT-qPCR). The results are presented as the mean ± SD of three independent experiments. B, E The mRNA levels of ME1 and GCLC/GCLM in OSCC and CRRR cells under normal conditions. C, F The mRNA levels of ME1 and GCLC/GCLM in OSCC cells and CRR cells transfected with si/Negative or si/Nrf2. At 48 h after transfection, total RNA was extracted, and the mRNA expression of Nrf2 was analyzed by RT-qPCR. G–I Nrf2-mediated-antioxidant capacity was examined using the GSSG/GSH quantification assay. G GSH production in HSC-2-WT and HSC-2-R cells 24 h after exposure to 10 Gy of radiation. GSH production in HSC-2-WT and HSC-2-R cells at 48 h after transfection with si/Negative or si/Nrf2. H At 24 h after irradiation, ROS/superoxide levels were examined in cells using a Cellular ROS/Superoxide Detection Assay Kit. I At 24 h after irradiation, ROS/superoxide levels were examined in cells transfected with si/Negative or si/Nrf2. The ROS/superoxide assay protocol is based on two fluorescent dyes: Oxidative Stress Detection Reagent (Ex/Em 490/525 nm) for the detection of total ROS and Superoxide Detection Reagent (Ex/Em 550/620 nm). *p < 0.05 and **p < 0.01.