Firefly luciferase-based reporter gene assays are the most commonly used assays 
to investigate the transcriptional regulation of gene expression. However, 
direct interaction of tested compounds with the firefly luciferase leading to 
altered enzymatic activity may lead to misinterpretation of experimental data. 
When investigating the proteasome inhibitors bortezomib, carfilzomib, and 
ixazomib, we observed increased luminescence for bortezomib and ixazomib, but 
not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which 
was inconsistent with the mRNA expression levels of the main PXR target gene 
CYP3A4. To further scrutinize this phenomenon, we performed experiments with 
constitutively expressed firefly luciferase and demonstrated that the increase 
in cellular firefly luciferase activity is independent from PXR activation or 
CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase 
enzyme, we made the counterintuitive observation that firefly luciferase 
activity is inhibited by bortezomib and ixazomib in a reversible and competitive 
manner. This inhibition stabilizes the firefly luciferase enzyme against 
proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life 
with subsequent enhancement of total cellular luminescence that eventually 
mimicked PXR-driven luciferase induction. These data show that particular 
compounds can strikingly interfere with firefly luciferase and once more 
illustrate the importance of careful interpretation of data obtained from 
luciferase-based assays.