Figure 7. E2F7 regulates the expression of ATX in human cancer cell lines.

(A) Schematic of 3C-qPCR primers targeting the MaeI restriction sites and TF binding sites in the human ENPP2 gene. (B) 3C-qPCR was performed in WT and p53-KO HCT116 cells using Anchor H (associated with the E2F7-RE2) and seven forward primers targeting E2F7-RE1 and promoter fragments. Relative interaction frequency was normalized to GAPDH and plotted as the mean ± SD. E2F7 siRNA was applied to (C) SKOV3 and (D) MDA-MB-231 cells. Cells were transfected with 150 nM of scramble or E2F7 siRNA for 24 h. Total protein was used for immunoblotting with antibodies against E2F7, ATX, and β-actin. Total cell lysates were fixed and subjected to ChIP-qPCR analysis with anti-E2F7 and nonspecific control IgG antibodies. Enrichment of E2F7-binding was normalized to the nonspecific control IgG. ChIP-qPCR was used to verify the interaction between E2F7 and ENPP2 promoter in the (E) SKOV3 and (F) MDA-MB-231 cells. (G) 3C-qPCR was applied to measure the interaction frequency between Anchor-H (associated with the intronic E2F7-RE2) and forward primers targeting E2F7-RE1 and promoter fragments in human SKOV3 and MDA-MB-231. The quantitative data are represented as the mean ± SD of three independent experiments. *p < 0.05, **p<0.01, and ***p < 0.001 indicate significant differences.