Cellular stress responses often involve changes in the stability of a critical regulatory protein, followed by downstream modulation of specific transcriptional targets. For many of these adaptive response pathways, the concentration and localization of a key transcription factor is tightly regulated. For example, under basal conditions the hypoxia-inducible factor-1A (HIF1A) transcription factor is kept at low levels by ubiquitin-mediated degradation. Under hypoxic conditions, ubiquitination is greatly reduced, leading to HIF1A accumulation in the nucleus and transactivation of various genes.
Our goal was to design a multiplexed assay enabling simple measurement over time of: 1) HIF1A protein accumulation; 2) HIF1A-mediated activation of transcription from the Hypoxia Response Element (HRE), and; 3) changes in cell viability due to compound treatment. Because of its small size and bright luminescence, NanoLuc® luciferase represents an ideal fusion partner for measuring changes in protein levels. The high sensitivity of NanoLuc® assays means that proteins can be expressed at lower, more physiological levels, yielding more biologically relevant results.
Dual-reporter assays provide a way to monitor multiple pathway points in the same population of cells. In a common experimental format, one luciferase is used as a reporter of transcriptional activation of a regulated promoter, while the other represents a negative control expressed from a constitutive promoter. Such a dual assay can improve data quality by allowing data normalization within the same well.
The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System allows sensitive and sequential monitoring of both NanoLuc® luciferase (Nluc) and firefly luciferase (Fluc) in the same sample using a simple add-read-add-read protocol. Efficient quenching of the initial Fluc signal coupled with the bright Nluc signal give high sensitivity to both reporters. If more data is desired from each treatment, the NanoDLR™ assay can be used to multiplex two experimental reporters, such as a NanoLuc® fusion protein with a firefly reporter gene assay.
Measurement of NanoDLR™ luminescence can be further multiplexed with a fluorescent measurement of cell viability using the CellTiter-Fluor™ Cell Viability Assay, for a more complete profile of the overall cellular response from a single sample. In this assay, a proteolytic activity associated with live cells converts a substrate into a fluorescent product that is proportional to the number of live cells. This assay can be performed immediately prior to addition of the lytic NanoDLR™ reagents.
The GloMax® Discover simplifies assay multiplexing and injector-based protocols, enabling easy time-course experiments.
Here is a sample protocol for measuring accumulation of HIF1A-Nluc and its subsequent activation of HRE-luc2P following treatment with the hypoxia mimetic, phenanthroline.
Materials to be Supplied by the User
Available from Promega:
Available from other Vendors:
- Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System (Cat.# N1610)
- GloMAX® Discover System (Cat.# GM3000)
- FuGENE® HD Transfection Reagent (Cat.# E2311)
- CellTiter-Fluor™ Cell Viability Assay (Cat.# E6080)
- pGL4.42 [luc2P/HRE/Hygro] Vector (Cat.# E4001)
- pNLF1-HIF1A [CMV/neo] Vector (Cat.# N1381)
- white, 96-well plates (Corning Cat.# 3917)
- 1,10-phenanthroline (Aldrich Cat.# 131377)
- HEK293 cells
- DMEM (Thermo Fisher Cat.# 11995065)
- Opti-MEM® I (Thermo Fisher Cat.#11058021)
- After dissociation, resuspension and centrifugation, resuspend HEK293 cells to 1.5 × 105 cells/ml in DMEM + 10% FBS.
- Create transfection complexes by mixing 20µg of pGL4.42 [luc2P/HRE/Hygro] Vector, 20ng of pNLF1-HIF1A [CMV/neo] Vector and 60µl of FuGENE® HD reagent in 2ml of Opti-MEM® I.
- Add the transfection complexes to 40ml of 1.5 × 105 HEK293 cells/ml, mix and dispense 72µl per well into 10 × 96-well plates. Incubate 18 hours in a 37°C + 5% CO2 incubator.
- Generate a serial dilution series of 10X concentrations of 1,10-phenanthroline in Opti-MEM® I to give the following final concentrations: 0, 0.1, 0.32, 1, 3.2, 10, 32 and 100µM. At time zero, add 8µl of a given 10X stock to the appropriate well (N=6) and place in a 37°C + 5% CO2 incubator.
- Prepare the ONE-Glo™ EX and NanoDLR™ Stop & Glo® Reagents. On the GloMax® Discover instrument, prime injector #1 with ONE-Glo™ EX Reagent and prime injector #2 with NanoDLR™ Stop & Glo® Reagent.
- At 0, 0.5, 1, 2, 3, 4, 6 and 8 hours after phenanthroline treatment, remove a given plate from the incubator, cool to room temperature and place in the GloMax® Discover.
- Perform the NanoDLR™ Injector protocol to inject both reagents, and measure luminescence in automated fashion (described in Technical Manual #TM426).
- To monitor for compound toxicity after 4 and 6 hours of compound treatment, add 20µl of CellTiter-Fluor™ Reagent (as a 5X reagent) to each well and incubate 1 hour in the 37°C incubator.
- Cool plates to room temperature and place in the GloMax® Discover. Perform the CellTiter-Fluor™ protocol (as described in Technical Bulletin #TB371, followed by the NanoDLR™ Injector protocol (Technical Manual #TM426).
Figure 1. Dose responses and time course following treatment of HEK293 cells with phenanthroline. Panel A. The dose response at a single time point displays phenanthroline-dependent accumulation of the HIF1A-Nluc fusion and a corresponding activation of the Hypoxia Response Element as measured by the reporter gene luc2P. Panel B. The fold-change in luminescence compared to untreated cells is plotted for both reporters over time following addition of 100µM phenanthroline. The time course highlights the lag that occurs between accumulation of HIF1A and its subsequent activation of transcription in the nucleus. Panel C. An additional cell viability measurement can be multiplexed with NanoDLR™ by incubating cells with CellTiter-Fluor™ and measuring fluorescence prior to performing the NanoDLR™ assay. In this case, the CellTiter-Fluor™ measurement indicates a small amount of toxicity at this time point at the higher concentrations of phenanthroline. Error bars represent S.E.M. for six replicates.
For many transcription factors in stress response pathways, regulated degradation is a key mechanism for controlling their activity. We have used the Nano-Glo® Dual Luciferase Reporter (NanoDLR™) Assay to multiplex a measurement of both protein accumulation with a NanoLuc fusion protein and the activity of the transcription factor on its regulated promoter with a luc2P reporter gene. In the case of HIF1A activity after treatment with phenanthroline, a time course demonstrates a lag between accumulation of the transcription factor and its activation of downstream targets. The brightness of NanoLuc® luciferase enables expression at physiologically relevant levels, which is important for observing the correct biological response.
Especially when NanoDLR™ is being used to measure two dynamic reporters, instead of having one constitutive control, it may be useful to include an assay for cell viability in order to monitor for toxicity caused by test compounds. This can be accomplished by multiplexing the luminescent assay with the fluorescent readout of CellTiter-Fluor™ Assay. The substrate is incubated with cells in media, and an enzyme associated with live cells converts this into a fluorescent product. The fluorescence can be measured on the GloMax® Discover instrument immediately before addition of NanoDLR™ reagents. The dual auto-injectors on the Discover can be used to automate the various reagent additions and measurements. This greatly simplifies the assay format described here, in which individual assay plates containing a compound dose response are removed from an incubator and placed into the luminometer at particular time points. These multiplexed measurements of protein abundance, gene expression, and cell viability can greatly increase the amount of information obtained from a single sample.