Journal: Molecular and Cellular Biology
Article Title: Nuclear Factor YY1 Inhibits Transforming Growth Factor ?- and Bone Morphogenetic Protein-Induced Cell Differentiation
Figure Lengend Snippet: YY1 represses TGF-β and BMP immediate-early gene responses. (A) YY1 represses PAI-1 induction by TGF-β1. Northern analysis of PAI-1 mRNA from Mv1Lu cells mock treated (−) or treated (+) for 24 h with 10 ng of TGF-β1 per ml in the absence of YY1 (−) or in the presence of increasing doses of YY1 (Ad-YY1) (triangle; MOI of 20 or 100) or control GFP (Ad-GFP) (MOI of 20 or 100) adenovirus is shown. Sample loading and blotting efficiency was monitored with β-actin mRNA on the same blot. (B) YY1 represses Id-1 induction by TGF-β1. Immunoblot analysis of endogenous Id-1 expression in MDA-MB-468 cells infected with the indicated MOI of recombinant adenoviruses and then treated with vehicle (−) or 2.5 ng of TGF-β1 per ml (+) for 1 h is shown. Arrows mark the positions of endogenous Id-1, adenoviral YY1, adenoviral Flag-Smad4 (F-Smad4), endogenous phosphorylated Smad2 (Smad2-P), and endogenous β-catenin, which serves as a loading and blotting efficiency control. (C) YY1 represses Id-1 induction by BMP-7. Immunoblot analysis of endogenous Id-1 expression in MDA-MB-468 cells is as for panel B but in response to 300 ng of BMP-7 per ml. Infection conditions were as for panel B, and arrows mark the same proteins as in panel B except for endogenous phosphorylated Smad1 (Smad1-P) instead of Smad2. Asterisks mark nonspecific protein bands. (D) YY1 represses PAI-1 promoter induction by TGF-β1. PAI-1 (800)-luciferase assays were performed in HepG2 cells with increasing amounts of YY1 plasmid (triangle; 0.1 to 1.0 μg) in the absence (−) or presence (+) of overnight stimulation with 2.5 ng of TGF-β1 per ml. The averaged data for normalized reporter activity are shown with standard errors derived from triplicate transfections repeated at least twice independently. (E) YY1 represses the Smad-specific 12xCAGA promoter. The assays were similar to those for panel D, with the 12xCAGA-luciferase reporter in HepG2 cells after overnight stimulation with 2.5 ng of TGF-β1 per ml. Data are reported as in panel D. (F) YY1 represses the Smad-specific Id1-BRE 2 promoter. The assays were similar to those for panel D, with the Id1-BRE 2 -luciferase reporter in C2C12 cells after overnight stimulation with 100 ng of BMP-6 per ml. Data are reported as in panel D.
Article Snippet: Murine C2C12 myoblasts, murine mammary epithelial NMuMG cells, mink lung epithelial Mv1Lu cells, monkey kidney COS-7 cells, human embryonic kidney 293T cells, human mammary MDA-MB-468 cells, and human hepatoma HepG2 cells were obtained and cultured according to protocols from the American Type Culture Collection.
Techniques: Northern Blot, Expressing, Multiple Displacement Amplification, Infection, Recombinant, Luciferase, Plasmid Preparation, Activity Assay, Derivative Assay, Transfection