Impact of cell culture conditions on NRF2 (nuclear factor E2 p45-related factor 2)-driven reporter gene expression

• The influence of growth medium, mechanotransduction, cycling phase, and culture dimensionality on ARE-luciferase reporter gene expression in HepG2 cells was investigated in vitro. • Basal and inducible luminescence readings, which indicate baseline and responsive NRF2 activity, differ across different cultivation conditions. • All tested conditions enable the identification of activated NRF2 signaling by the test compounds. For the identification and characterization of compounds that modulate the activation status of the stress-responsive and cytoprotective transcription factor NRF2 (nuclear factor E2 p45-related factor 2), ARE (antioxidant response element)-driven reporter gene assays serve as convenient tools. NRF2 signaling is susceptible to various factors, including cellular energy status, circadian rhythm, and mechanical or oxygen tension. These parameters are often inadequately accounted for in routine 2D cell culture and screening processes, potentially limiting the relevance of the obtained data or identified hits. Therefore, we investigated whether NRF2-driven luminescence readings from a ARE-luciferase reporter gene markedly differ from routine culture conditions when stably transfected HepG2 cells are cultivated in plasma-like medium, exhibit altered mechanotransduction, are synchronized, or grown in spheroids. While NRF2 signaling is consistently activated by the synthetic triterpenoid CDDO-IM under all tested conditions, the baseline (indicative for the initial cellular stress status/Nrf2 activity) and/or the extent of inducible luciferase activity (activation amplitude conferred by the NRF2 activator) varies across different cultivation conditions.