Fig. 1
(A) Schematic representation of the BRET-based redox-sensitive biosensor ROBINy in both oxidized and reduced states (adapted from Fu et al.30). ROBINy consists of nanoLuc ΔN4 (nluc lacking the N-terminal 4 amino acids) and Re-Qy ΔC12 (a redox-sensitive fluorescent acceptor lacking the C-terminal 12 amino acids). Under oxidative conditions, disulfide bonds form within Re-Qy, increasing its fluorescence and thus enhancing the energy transfer from nluc to Re-Qy, resulting in a higher BRET ratio. (B–D) Timeline of experiments conducted in XP6BE human fibroblasts transfected with cytoplasmic or mitochondrial ROBINy. B) Direct ROS response assay: cells were sham- or RF-EMF-exposed (5G-modulated, 3.5 GHz) for 24 h. During the last 10 min of exposure, cells were treated with either H₂O₂ (0–100 mM) or Kp372-1 (0–100 µM). Alternatively, cells were treated with Antimycin A (0–300 µM) during the final 4 h of the exposure period. (C) Positive control for adaptive response: cells were pretreated with arsenic trioxide (As₂O₃, 3 µM, 20 h; adaptive dose, AD), then challenged with increasing concentrations (challenging dose, CD, 30 min). (D) RF-induced adaptive response assay: cells were exposed to RF-EMF or sham-exposed for 20 h, rested for 3 h, then challenged with CD of As₂O₃ for 30 min. BRET was measured immediately after the end of the RF exposure and/or chemical treatment.
