Fig. 2.

KBP gene is negatively regulated by T₃. A, Diagram of mouse KBP gene and reporter construct basal expression, response to T₃, and fold T₃ repression (basal/T₃ treated). B, Transfection assays. HepG2 cells grown in serum-free medium were cotransfected with reporter construct and TRβ expression vector. The fragment −59/+723 includes nTRE1 (contained in −59/+66) and nTRE2 (contained in 93/135). Cells were treated with T₃ to a final concentration of 0, 5, 50, or 100 nm for 24 h before luciferase assay. The dual-luciferase assay system was used to determine relative Firefly luciferase activity (FLuc)/Rhenilla luciferase (RhLuc) of the reporters. Values shown are mean ± se (error bars) of triplicates. *, P < 0.05, **, P < 0.01, when compared with conditions without TRβ or added T₃. C, ChIP analysis of TR binding to nTRE1 −59/+66 and nTRE2 93/135. The segment 254/431 is a control fragment without a known TRE or nTRE. ChIP was performed in GC-1spg cells transfected with TRβ.