Oxidative Stress and Hypoxic Responses
NanoLuc® luciferase is ideally suited as a protein function reporter for quantifying protein lifetime dynamics due to its small size (19 kDa) and intense bioluminescence output. Changes in intracellular protein levels can be quantified using Nano-Glo® reagent in a simplified assay format, wherein the luminescence output of NanoLuc luciferase is used as a surrogate for the intracellular levels of the fusion protein. It is possible to measure hypoxia or oxidative stress-response signaling via direct genetic fusions of NanoLuc luciferase with HIF1A or NRF2 proteins, respectively, in HCT116 colorectal cancer cells.
As shown in Figure 1, when HCT116 cells were transiently transfected with HIF1A-NanoLuc plasmid DNA, a chemical hypoxia mimetic (1,10-phenanthroline) induced a dose- and time-dependent accumulation of the reporter fusion. Similarly, an NRF2-NanoLuc fusion protein accumulated in HCT116 cells upon treatment with d,l-sulforaphane (a known inducer of reactive oxygen species).
Although protein stabilization is an initiating event in stress-pathway activation, a key outcome is the subsequent induction of genes required for maintaining cellular homeostasis. Multiplexed analysis of a NanoLuc protein fusion reporter with a traditional firefly luciferase transcriptional reporter allows for both signaling events to be analyzed in a single sample.
As an example, HEK293 cells were co-transfected with HIF1A-NanoLuc and hypoxia response element (HRE-luc2P) reporters, and the Nano-Glo Dual-Luciferase® Reporter Assay was used to sequentially assess both protein stability and transcriptional responses from the same cell population.