Figure 3. NCoR is involved in transcriptional inhibition of the VEGF-C and VEGF-D genes by TRβ.

(A) Transient transfection assays in MDA and MDA-TRβ cells with reporter plasmids of the human VEGF-C promoter extending to nucleotides −1059 and −201 or the empty plasmid without promoter sequences (e.p). Luciferase activity (means ± S.D) was determined in cells treated for 36 h in the presence and absence of 5 nM T3 and is expressed relative to value obtained in the untreated cells transfected with the empty plasmid. Differences between untreated and T3-treated cells were calculated with the t-test and are indicated as ***P < 0.001. (B) similar experiments in cells cotransfected with the −201 plasmid and control or NCoR siRNAs. Luciferase activity (means ± S.D) was measured in cells treated with and without T3 and is expressed relative to that obtained in untreated MDA cells transfected with siControl. Statistically significant differences of the ANOVA post-test among groups of MDA and MDA-TRβ cells are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001. (C) ChIP assays with the indicated fragments of the VEGF-C and VEGF-D promoters and NCoR and TRβ antibodies in cells treated in the presence and absence of T3 for 1 h. Schemes of the promoter fragments used showing the existence of putative binding sites for TR (TRE hemisites) and for other transcription factors are depicted. Results are expressed as % of the input after subtracting the values obtained after immunoprecipitation with control IgG that were always lower than 1% of the input. Data shown are the mean of two independent experiments.