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. 2009 May 28;4(5):e5698. doi: 10.1371/journal.pone.0005698

Figure 4. Promoter luciferase assay indicates regulation of β-catenin and GATA4 by NKX2-5.

Figure 4

(A) The graphs demonstrate promoter activity luciferase reporter assay. For this assay, the regions between primers GF2 and GR2 for GATA4 and BF2 and BR2 for β-catenin were amplified and cloned into pGL3-promoter plasmid. Luciferase reporter assay using these constructs demonstrated repression for β-catenin sequence, and activation for GATA4 sequence, of promoter activity when the adenovirus expressing NKX2-5 was co-transfected into COS7 cells. Cells transfected with the constructs were exposed to various MOIs (5-40 MOI) of Nkx2-5-adenovirus as indicated (n = 3 for each concentration). (B) Mutational analysis of β-catenin and GATA4 promoters. The approximate positions of mutated NKEs in β-catenin and GATA4 promoters and deletion of the 5′ half (mNKEΔ) of the β-catenin promoter sequence have been indicated. Luciferase reporter assay was performed using wild type (WT-B: β-catenin, and WT-G: GATA4) and mutated (mNKEs) promoters after adding adenovirus expressing NKX2-5 at either 15 (gray bars) or 30 (black bars) MOI. White bars indicate promoter activities in untreated cells. Activities of the promoters harboring mutated NKEs were all statistically different from the equally treated WT values (# and * indicate P<0.02) except for deletion of sequence not containing an NKE (mNKEΔ10).