PXR-mediated regulation of TLR4 signaling is not directly dependent on TLR4 gene transcription but instead through regulating TLR4 mRNA stability. (a) Nuclei of LS174T cells were isolated at various time points after treatment with the human PXR ligand rifampicin (10 μM) and used in the nuclear run-on transcription reaction. After the reaction, biotinylated RNA was purified and used for cDNA synthesis and real-time qPCR. The transcription efficiency of TLR4 and classical PXR target gene, MDR-1, were analyzed relative to that of β-actin. (b) Actinomycin D (20 μg/ml) chase experiment was performed in LS174T cells treated with or without rifampicin to evaluate TLR4 mRNA stability. After rifampicin treatment for 48 h, TLR4 mRNA half-life decreased from ~243 to ~127 min. PXR, pregnane X receptor; TLR4, Toll-like receptor 4.