Figure 1
T 3 -regulates BSSP4 mRNA and protein expression in HepG2 cells. (A) Expression of TR in cell extracts of overexpressed-TR and parental cell lines was determined via Western blotting. The positions of 47 kDa TRα1 and 55 kDa TRβ1 are indicated. BSSP4 expression was determined in the three stable HepG2-TR lines and HepG2-neo cells at 12–48 h in the absence or presence of 1 and 10 nM T3 using (B) Q-PCR and (C) Western blotting. (D) T3 stimulated BSSP4 expression in J7 and Huh7 cells expressing endogenous TR was determined by Western blotting. (E) HepG2-TRα1 cells were transfected with the luciferase reporter plasmid driven by the BSSP4 5′-flanking region (positions -2066 to -7 containing twelve putative TRE sites) with or without pA3TK-luc. Promoter activities were calculated, relative to 0 nM T3 (+T3/-T3), and further normalized to the pA3TK-luc control as well as β-galactosidase activity (T3-induced changes were normalized to that of β-gal). Columns, mean values obtained from at least three independent experiments performed in triplicate; bars, SE. (F) ChIP assay demonstrating that TR is recruited to the BSSP4 5′-flanking region, together with RXR in HepG2-TRα1 and J7-TRα1 cells. Two sets of primers for BSSP4 TRE, positive control TRE (FURIN) and negative control (GAPDH) were prepared.