Fig. 5.

Knockdown of hPXR expression attenuates DIM-induced hPXR target gene expression in HepG2 and LS174 T cells. (A) HepG2 cells stably expressing FLAG-hPXR were transfected with control non-silencing siRNA or hPXR siRNA. Whole-cell lysates were collected 72 h after siRNA transfections and subjected to western blot analysis using anti-FLAG and anti-actin antibodies (as a loading control). COS7 cells transiently transfected with FLAG-hPXR or FLAG-pcDNA served as positive or negative controls, respectively, for hPXR expression. Data shown are from a representative experiment. (B) 48 h after siRNA transfections, the cells were transiently cotransfected with CYP3A4-luc and CMV-Renilla (transfection control) plasmids, and 24 h after transfection, the cells were treated for another 24 h with DMSO, RIF, or DIM, as indicated. The firefly and renilla luciferase activities were measured 24 h after the treatments using Dual-Glo luciferase assay system. The normalized CYP3A4 promoter activity is expressed as relative luciferase units and presented as fold change over the DMSO control. Data represent mean ± SD from three independent experiments. Statistical significance (*, p < 0.05) was determined using Students t test by comparing hPXR siRNA samples with control siRNA in each drug treatment group. (C, D & E) LS174 Tcells were transiently transfected with hPXR siRNA or control non-silencing siRNA. 48 h after transfection, the cells were treated for another 24 h with DMSO, RIF or DIM, as indicated, and mRNA expression of hPXR (C), CYP3A4 (D) and MDR1 (E) was determined. The induction of each gene mRNA level by the treatment was normalized as fold increase over the untreated control. Data represent mean ± SEM from three independent experiments. Statistical significance (*, p < 0.05) was determined using Students t test by comparing hPXR siRNA samples with control siRNA in vehicle or each drug treatment group.