Figure 6. DNMT1 inhibits TP53BP2 expression by inversely modulating E2F1 in trophoblasts. (A) E2F1 expression in placentas was detected via qRT‒PCR and western blotting. (B) Immunofluorescence staining was used to detect E2F1 expression (green) in trophoblasts of the placenta. Scale bar=50 µm. (C) TP53BP2 expression was detected via western blotting in HTR8/SVneo cells transfected with Ad-E2F1 or sh-E2F1 under hypoxia. (D) The enrichment of E2F1 at the promoter region of TP53BP2 in HTR8/SVneo cells under hypoxia was examined via a ChIP assay with an E2F1 antibody. (E) A schematic diagram of the predicted E2F1-binding sites in the TP53BP2 promoter region from the JASPAR database (http://www.genereg.net/). The blue box represents the -33/-22 site. The green box represents the -99/-88 site. The yellow box represents the -368/-357 site. (F) A ChIP assay was conducted to assess the binding of E2F1 to specific sites (-33/-22, -99/-88, and -368/-357) on the TP53BP2 promoter in HTR8/SVneo cells under hypoxia. (G) The promoter activities of TP53BP2 with the wild-type (WT) or mutant -33/-22 site (Mut1), -99/-88 site (Mut2) and -368/-357 site (Mut3) of E2F1 were determined via a luciferase reporter assay in HEK293T cells. (H) Co-IP assay followed by immunoblotting showing the interactions between DNMT1 and E2F1 in HTR8/SVneo cells under hypoxia. (I) A ChIP assay was performed to demonstrate that E2F1 binds to the TP53BP2 promoter in HTR8/SVneo cells transfected with sh-DNMT1. The data are presented as the means ± SDs. **P <0.01,***P <0.001.