FIG. 2.

(A) Activation of a Stat1-responsive reporter by the Stat1-ER chimera in Stat1-deficient U3A cells. U3A cells were transfected with an empty expression vector (pcDNA3.1) or an expression vector for either Stat1-ER or wtStat1 plus the Stat1-responsive reporter IRF-1x4-tk-luc and a β-Gal control plasmid. After transfection, cells were treated with estrogen (E₂; 20 h), tamoxifen (Tam; 20 h) or IFN-γ (5 h), lysed, and assayed for luciferase and β-Gal activities. The normalized responses were determined by dividing relative light units measured in a luciferase assay with the β-Gal activity in the same lysate, and the fold induction (induced/untreated) was calculated by using the averaged normalized responses from three independent experiments. (B) HepG2 cells were transfected with the IRF-1x4-tk-luc reporter and an expression vector for Stat1-ER (circles) or the estrogen-responsive ERE-tk-luc reporter and an expression vector for wild-type ER (diamonds). Both sets included a β-Gal control plasmid. Cells were treated with the indicated concentrations of estrogen (20 h), lysed, and assayed for luciferase and β-Gal activities. The normalized responses are expressed as a percentage of the maximal normalized response for each treatment and are presented as the mean of three independent experiments. (C) Effect of Stat1 Ser⁷²⁷ Ala mutation on transcriptional activity of Stat1-ER and wtStat1 in Stat1-deficient U3A cells. (D) DNA sequence specificity of the Stat1-ER chimera. Stat1-deficient U3A cells were transfected with an expression vector for Stat1-ER and either a Stat1-responsive reporter (IRF-1x4-tk-luc) or a Stat6-selective reporter (mGɛx4-pGL2) together with a β-Gal control plasmid. Treatments and calculations were performed as described for panel A. un, untreated; Tam, tamoxifen treated.