Jing manuscript original data_ Visualization of CYP3A4 Expression at both mRNA and Protein Levels Simultaneously at Single Cell Resolution in Various Conditions Using RNAscope Combined with Immunofluorescence

Research Hypothesis and Summary

We hypothesized that CYP3A4 expression is dynamically regulated at both mRNA and protein levels during hepatic differentiation and under various treatment conditions, and that a combined RNAscope and immunofluorescence (IF) approach would enable simultaneous, spatially resolved detection of CYP3A4 transcription and translation at single-cell resolution.

To test this, we employed a dual-modality imaging method using RNAscope for mRNA detection and IF for protein visualization in HepaRG cells across four time points (day 7, 14, 21, 28) and under five treatment conditions (glucose, FFAs, rifampicin, APAP, control). Two quantification strategies were used: whole-field intensity to mimic bulk assays (RT-qPCR, WB, FC, ELISA), and cell-specific intensity in CYP3A4-positive cells.

Key Findings

Cell Model Comparison: Among three hepatic models, only differentiated HepaRG cells robustly expressed CYP3A4 in HLCs, with expression absent in CLCs, HepG2, and Huh7 cells.

Differentiation Dynamics: CYP3A4 mRNA levels gradually increased in whole-field measurements but did not show significant differences between adjacent time points. Protein levels significantly increased from day 14 onward. In positive cells, mRNA increased progressively through day 28, while protein levels plateaued after day 14.

Single-Cell Patterns: Five distinct CYP3A4 expression patterns were identified, reflecting different stages from transcription initiation to translation and downregulation. These patterns revealed asynchronous mRNA-protein expression in individual cells.

Treatment Effects:

Glucose (50 mM): No effect on mRNA or protein levels.

FFAs (500/1000 µM): Both doses reduced CYP3A4 mRNA and protein levels; higher FFA caused a greater reduction in mRNA.

Rifampicin (10 µM): Significantly increased mRNA, but not protein levels.

APAP (10 mM): Decreased both mRNA and protein, with visible loss of CYP3A4-positive HLCs likely due to selective cytotoxicity.

Interpretation and Application

This study demonstrates that CYP3A4 expression is regulated in a cell-type- and condition-specific manner, with transcription and translation not always aligned. The combined RNAscope-IF technique allows dual detection of CYP3A4 mRNA and protein within the same cells, offering both spatial and quantitative insights. This platform provides a valuable tool for studying hepatic functional heterogeneity, xenobiotic metabolism, and drug-induced toxicity at the single-cell level, and can inform preclinical drug screening, toxicology, and pharmacokinetic modeling.