Fig. 7. Effect of GRIM-19 on the transcriptional activity of Stat3 and target gene expression. (A–C) COS-1 and MCF-7 cells were co-transfected with a vector control, Stat3 and/or GRIM-19, the truncation mutant GRIM-19_1–101 or GRIM-19_36–144 together with a reporter plasmid pSIE-CAT and pCMV-β-gal. Cells were either left uninduced or stimulated with EGF (100 ng/ml) for 6 h in (A) or IFN-β and RA for 24 h in (B). Cells were harvested and the lysates used for CAT assays were normalized with equivalent β-galactosidase activity, and CAT assay was performed as described (Jain et al., 1998). The averages of the relative CAT activities from three independent experiments are shown in the graph, with error bars representing the standard deviation of the means. (D and E) GRIM-19 inhibits α₂-macroglobulin report expression. HepG2 cells were co-transfected with pGL3-α2M-215luc or the empty luciferase vector (pGL3), GRIM-19, antisense GRIM-19 plasmid or the empty vector as labeled, together with pRL-TK for normalization. Cells were left non-stimulated (–) or were induced overnight with IL-6 (+). Relative luciferase values are indicated, which represent the means of six samples with the standard deviation given as the error bars. Cells were pre-treated for 55 h with IFN-β (500 U/ml) and RA (1 µM) before stimulation by IL-6 in (E).