Fig. 4

TFAP2C binding site regulates luciferase reporter activity and TFAP2C negatively regulates MMP9 expression. (a) A TFAP2C binding site was identified in HD11. The TFAP2C binding motif is from the JASPAR database⁴⁴. (b) The TFAP2C binding site shown in (a) was deleted from the E1-pGL4.23 vector (TFAP2C-del), and luciferase assays showed significantly higher activity than E1. The y-axis shows luciferase activity relative to the empty pGL4.23 vector. (c) TFAP2C gene expression was significantly decreased after treatment with both siRNA#1 and siRNA#2. TFAP2C siRNA#1 (blue) did not significantly increase MMP9 expression, and TFAP2C siRNA#2 (pink) treated cells show a significant increase in MMP9 gene expression. (d) Images and (e) quantification of western blots showing that TFAP2C protein expression was significantly decreased after treatment with both siRNA#1 and siRNA#2. MMP9 protein expression was significantly increased in TFAP2C siRNA#1 and siRNA#2 treated cells. In (c) and (e), the y-axis shows the log₂-transformed fold change values compared to the negative control siRNA treatment, for gene expression and western blot quantification respectively. The black dots in (b), (c) and (e) are individual biological replicate values for the experiment. Details of antibody concentration and cell counts used for western blots are provided in Supplementary Table S9. n.s. indicates not-significant, (*) indicates p-value < 0.05, (**) indicates p-value < 0.01, (***) indicates p-value < 0.001. All experiments were performed with three biological replicates. Results are displayed as the mean +/- standard error (SE), and p-values were calculated using a two-tailed student’s t-test.