Visualization of CYP3A4 expression at both mRNA and protein levels simultaneously at single cell resolution in various conditions using RNAscope combined with immunofluorescence.

Precise characterization of drug-metabolizing enzyme expression, such as CYP3A4, at the single-cell resolution is essential for understanding hepatic functional heterogeneity and its impact on xenobiotic clearance. In this study, we employed RNAscope in situ hybridization combined with immunofluorescence to simultaneously visualize CYP3A4 mRNA and protein expression in individual cells under diverse experimental conditions. Using differentiated HepaRG cells, we identified distinct CYP3A4-positive and CYP3A4-negative subpopulations in metabolic zonation. Nonconfluent HepG2 and Huh7 cells showed nearly undetectable CYP3A4 mRNA and protein signals. Five distinct CYP3A4 expression patterns were identified in differentiated HepaRG cells, capturing various stages of mRNA and protein production and reflecting the stepwise progression of gene expression from transcription to translation at single-cell resolution. CYP3A4-positive cells showed dynamic changes during growth and differentiation stages in HepaRG cells. Metabolic stressors, such as free fatty acids, significantly downregulated CYP3A4 expression in steatotic HepaRG cells, identified by lipid droplet morphology. Hepatotoxicity inducer acetaminophen resulted in significant cell death and downregulated CYP3A4 expression, sensitive only to CYP3A4-positive cells but not to CYP3A4-negative cells. Exposure of rifampicin to differentiated HepaRG cells induced CYP3A4 at both transcriptional and translational levels, but only in CYP3A4-positive cells, not CYP3A4-negative cells. In conclusion, the combined RNAscope-immunofluorescence approach enabled analysis of cell type-specific and condition-dependent regulation of CYP3A4 at single-cell resolution, revealing transcription-translation discordance and metabolic zonation-like effects. This approach provides a powerful tool for studying hepatic drug metabolism and toxicity in heterogeneous liver models. SIGNIFICANCE STATEMENT: This study introduces a powerful dual-modality imaging approach that enables simultaneous visualization of CYP3A4 mRNA and protein at single-cell resolution with heterogeneous hepatic cultures. By integrating RNAscope with immunofluorescence, this study uncovers how metabolic stressors and drug inducers differentially modulate CYP3A4 expression across distinct liver cell subtypes. This platform enhances the ability to study cell type-specific drug metabolism, offering critical insights into hepatic functional heterogeneity.