FIG. 1.

Regulation of pBi-L by doxycycline and thyroid hormone (T₃). (A) From left to right, the pBi-L plasmid itself (pBi-L), the same vector with a TRα1 insert, the pBi-L plasmid together with a pSG5-TRα1 expression vector, the pBi-L vector with a TRβ1 insert, or the pBi-L plasmid together with a pSG5-TRβ1 expression vector were transiently transfected into Hepa 1-6 cells using the lipofection procedure previously described (1). Transfections also included a pCH110 β-galactosidase construct as an internal normalization control (1). Cells were maintained at 37°C in Dulbecco-modified Eagles medium containing 10% hormone-depleted fetal bovine serum. After 24 hours the transfection medium was replaced with medium containing doxycycline, T₃, or both, as indicated below each panel. After an additional 24 hours the cells were harvested and relative luciferase activity was calculated (1) and is presented. (B) The overall transfection protocol in panel A was repeated using either the TRα1-pBi-L construct described above, a related (TRα-pUHD10-3) construct based on a similar plasmid lacking the luciferase gene (pUHD10-3), and either Hepa 1-6 cells or HepG2 cells as indicated. After harvesting the cells, mRNA was isolated and quantified for levels of TRα1, luciferase, and GAPDH using a reverse transcription PCR protocol (4). (C) Alternatively, the pBi-L vector, a linearized version of the pBi-L vector (lin), or a fragment of the same construct limited to the luciferase-bidirectional promoter region (tetO-Luc) were tested together with the TRa expression vector in the same transfection/luciferase assay as described in panel A.