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. 2011 May 20;12(7):665–672. doi: 10.1038/embor.2011.78

Figure 5.

Figure 5

TIF1γ inhibits TGF-β1 signalling. (A) HMEC-TR cells inactivated for TIF1γ or Smad4 were treated with TGF-β1 for 9 h. TGF-β1-induced fold changes of PAI1 were analysed by qRT–PCR. All values were normalized to the amount of HPRT messenger RNA and expressed relative to the value obtained for TGF-β1-untreated controls. The experiment shown is representative of three separate experiments performed in triplicate; error bars represent s.d. (B) HMEC-TR cells were co-transfected with the pGL3(PAI1)-Luc vector together with the pRL-CMV (cytomegalovirus) internal control vector and expression vectors or short-interfering RNA, as indicated. Relative luciferase activity is given as the mean±s.d. of an experiment performed in triplicate, representative of three experiments. (C,D) ChIP assay was performed on HMEC-TR cells transfected (D) or not (C) with TIF1γ expression vector as indicated and treated with TGF-β1 for 2 h. PCR amplification of endogenous PAI1 promoter (733/484) was performed to detect protein-bound DNA. Rabbit preimmune serum was used as a negative control. Primers specific to PAI1 were used before (input) and primers specific for actin were used after immunoprecipitation as a control to monitor immunoprecipitation specificity. (E,F) ChIP assay of the kinetics of Smad4 and TIF1γ binding to DNA after TGF-β1 stimulation. HMEC-TR cells transfected with PAI1 promoter were subjected to PCR analysis (E). The relative binding of Smad4 and TIF1γ proteins to the human PAI1 promoter was also measured by qRT–PCR analysis of the precipitated DNA and input DNA (F). Results are expressed as percentages of the total DNA input. The data shown correspond to one of two independent experiments performed with comparable results. (G) ChIP assay of the kinetics of Smad4 binding to DNA after TGF-β1 stimulation. HMEC-TR cells transfected with PAI1 promoter and inactivated for TIF1γ were subjected to PCR analysis. AU, arbitrary units; ChIP, chromatin immunoprecipitation; ctrl, control; IgG, immunoglobulin G; IP, immunoprecipitation; PAI1, plasminogen activator inhibitor 1; qRT–PCR, quantitative reverse transcription PCR; S4, Smad4; TGF-β1, transforming growth factor-β1; TIF, transcription intermediary factor 1γ.