Fig. 3

Monitoring degradation of Dendra2-Nrf2 under basal and induced conditions in single live cells. HepG2 cells (5 × 10⁵) stably expressing Dendra2-Nrf2 (A) or Dendra2 (C) were seeded on glass bottom MatTek dishes for 48 h later. Prior to visualization, some cells were pre-treated with tBHQ (20 µM) for 2 h or MG-132 (10 µM) before photoconversion. Single cells expressing Dendra2- Nrf2 or Dendra2 were demarcated around the cell margin by the auto-detection feature of NIS Elements Advanced Research 4.0 software and photoconverted using a 405 nm laser for 2.0 sec to the indicated area. Representative image panels of single cells expressing Dendra2-Nrf2 or Dendra2 under basal conditions (no treatment) is illustrated in panels A and C, respectively. Time lapse images were captured every 45 seconds in the green and red channels after photoconversion and processed as described in materials and methods. Any change(s) in morphology and position of the cells over time was adjusted by the imaging instrument using NIS Elements Advanced Research 4.0 software. The average fluorescence intensity for Dendra2-Nrf2 (n= 5–7) or Dendra2 (n= 5–22) was plotted over time using one-phase decay analysis of GraphPad Prism 7 with error bars representing the standard deviation. Scale bars, 5 μm.