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. 2011 Jul 19;6(7):e22129. doi: 10.1371/journal.pone.0022129

Figure 5. DKK1 is a GATA6 target gene.

Figure 5

(A) Venn diagram indicating the number of dysregulated genes identified by microarray analysis of AsPC1-GATA6sh (left circle, blue) and A13A-GATA6sh cells (right circle, yellow). The green cross-area indicates commonly dysregulated genes and includes DKK1. (B) Real-time PCR confirming DKK1 overexpression in AsPC1-GATA6sh and A13A-GATA6sh cells. (C) Detection of secreted DKK1 protein in conditioned media in AsPC1-GATA6sh and A13A-GATA6sh cells. Secreted DKK1 protein levels are indicated by using absorbance OD450. (D) Chromatin immunoprecipitation assay confirming binding of GATA6 to the DKK1 promoter. Non-immune IgG and whole genome derived gDNA are used as negative and positive controls, respectively. (E) EMSA assay confirming binding of GATA6 to putative GATA binding sites #2 and #3. The mutant sequence mGATA-#3 did not generate any detectable binding. Nuclear Extr, nuclear extract; GATA6-P, a positive control probe derived from the TFF2 promoter containing a GATA6 binding site; GATA-#2, probe containing putative GATA binding site No. 2; GATA-#3, probe containing putative GATA binding site No. 3; mGATA-#3, probe containing a mutated putative GATA binding site No. 3; refer to methods for additional details (F) Effect of GATA6 expression on activity of the DKK1 promoter. Data is presented as the ratio of firefly luciferase activity to sea pansy luciferase activity. pGL3 was used as a negative control for background. (G) Real-time PCR for DKK1 mRNA expression in Panc1 cells. When appropriate, all experimental data shown represents the summary three independent experiments. *, p<0.05; ***, p<0.001.