A, Dio2 gene is induced by C/EBPs: luciferase assay of 1299 ATG Dio2 luc promoter construct in JEG3 cells. Cells were seeded in six-well plates and transfected with 1299 ATG Dio2 Luc, RSV β-gal, C/EBPα, and C/EBPβ plasmids. At the end of the incubation, luciferase and β-galactosidase assays were performed. ANOVA test: *, C/EBPα vs. control, P < 0.05; **, C/EBPβ vs. control, P < 0.001. Results represent the average value ± sd of three different experiments, each performed in triplicate. B, Endogenous expression of Dio2 mRNA in JEG3-C/EBPα and JEG3-C/EBPβ stable cell lines; top, Western blot (WB) with C/EBPα and C/EBPβ antisera; bottom, RT-PCR analysis, using primers specific for Dio2 and β-actin cDNA. C, Stability of C/EBP-induced transcript. C/EBPβ stable cell line (JEG3β) cells were incubated with 5 μg/ml actinomycin D for the indicated times. At the end of the incubation, total RNA was extracted and reverse transcribed, and the cDNA was amplified with primers complementary to Dio2 and β-actin genes (top); bottom, densitometric analysis of the gel. D, Effect of siRNA-mediated knockdown of C/EBPs on Dio2 mRNA expression; top, quantitative RT-PCR of RNA extracted from JEG3 cells transfected with control (siCtr) and C/EBPα and C/EBPβ siRNA, with Dio2 mRNA levels normalized to GAPDH mRNA levels; bottom, Western blot with C/EBPα, C/EBPβ, or tubulin antisera to show the efficacy of siRNA-mediated knockdown. ANOVA test: *, C/EBPα vs. control, P < 0.001; **, C/EBPβ vs. control, P < 0.001. Results represent the average value ± sd of three different experiments, each performed in triplicate.