Fig. 3.

PXR represses FoxA2 trans-activation of the mouse Cpt1a promoter in Huh7 cells. A. Reporter plasmid, pGL3/CPT1A -2.4k was co-transfected with or without FoxA2 in the presence or absence of mPXR and hRXR as indicated. At 24 h after the transfection, cells were treated with DMSO or PCN and incubated for additional 24 h. Relative luciferase activities were calculated by taking the activity of the DMSO treated cells transfected with the reporter plasmid alone as one. B. A series of pGL3/CPT1A reporter plasmids as indicated were co-transfected with or without FoxA2 for 48 h. Numbers on the vertical ax indicate lengths of the Cpt1a promoter cloned into the reporter plasmids. Relative luciferase activities were calculated by taking the activity of the cells transfected with the pGL3/CPT1A -2.4k without FoxA2 as one. C. In vitro-translated FoxA2 was incubated with ³²P-labeled double-stranded putative FoxA2 binding sites, C1, C2 and C3 within the Cpt1a promoter and FoxA2 binding site of IGFBP-1 promoter. Cold probes stand for the one hundred-fold molar excess competitor co-incubation. Shifted bands were separated by electrophoresis on a 4% polyacrilamide gel. Bands were detected by autoradiography. D. Reporter plasmid, pGL3/CPT1A -2.4k and the deletion mutants of the putative FoxA2 binding sites, C1 and C3, were co-transfected with or without FoxA2 for 48 h. Relative luciferase activities were calculated by taking the activity of the cells transfected with the pGL3/CPT1A -2.4k without FoxA2 as one. Bars indicate mean ± standard deviations in A, B, and D.