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• W4281671693 abstract "The activity of nuclear receptors (e.g., pregnane x receptor, PXR) can be assessed by luminescence-based dual reporter gene assays. Under most conditions, receptor-activated firefly luminescence is normalized to Renilla luminescence, which is triggered by a constitutively active promoter. Simultaneous damage to the cells can however disrupt these signals and thus impair the interpretation of the data. Consequently, this study addressed three important aspects: First, idealized models were described, each highlighting crucial characteristics and important pitfalls of dual PXR reporter gene assays used to evaluate PXR activation or inhibition. Second, these models were supported by experimental data obtained with a strong PXR activator (rifampicin) with low cytotoxicity, a PXR activator with high cytotoxicity (dovitinib), a proposed PXR inhibitor that reportedly has no toxic effects (triptolide), and a cytotoxic control (oxaliplatin). Data were evaluated for relative PXR activity data, individual firefly or Renilla luminescence, and anti-proliferative effects of the compounds (assessed by crystal violet staining). Finally, a step-by-step guide is proposed to avoid misleading set-up of the assay or misinterpretation of the data obtained. Key considerations here include (1) omission of drug concentrations beyond 10-20% proliferation inhibition; (2) observation of Renilla luminescence, because this tends to indicate 'false PXR activation' when it inexplicably decreases; (3) parallel decrease of relative PXR activity and proliferation below baseline levels in conjunction with a sharp decrease in Renilla luminescence indicates 'false PXR antagonism'; (4) non-sigmoidal relationships suggest the absence of concentration dependency." @default.
• W4281671693 created "2022-06-13" @default.
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• W4281671693 date "2022-06-09" @default.
• W4281671693 modified "2023-09-25" @default.
• W4281671693 title "How to avoid misinterpretation of dual reporter gene assay data affected by cell damage" @default.
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• W4281671693 doi "https://doi.org/10.1007/s00204-022-03323-0" @default.
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