BAZF down-regulates Notch signaling by targeting CBF1. (A-B) Disruption of network formation by BAZF knockdown was rescued by DAPT, Notch1 siRNA, or CBF1 siRNA. (A) HUVECs were transfected with indicated combinations with siRNAs and DAPT. After the transfection and serum-starvation, the cells were seeded on the Matrigel with or without DAPT (1μM) in the presence of VEGF-A. Scale bar: 100 μm. (B) Total network length per mm² was quantified by ImagePro Plus. The results represent mean values from 3 experiments. Error bars represent ± SD. (C) BAZF knockdown increases the expression of Notch target genes, HEY1 and HEY2. The expression levels of the HEY1, HEY2, VEGFR2, and BAZF mRNAs were analyzed by quantitative RT-PCR. (D) BAZF overexpression down-regulates HEY1 and HEY2 expression. The expression levels of HEY1 and HEY2 mRNAs were analyzed by quantitative RT-PCR (*P < .05, ***P < .001). (E) Interaction of BAZF with CBF1. The cell lysates were prepared from VEGF-A–treated (6 hours) or non-treated HUVECs and immunoprecipitated with an anti-CBF1 antibody. The immunoprecipitates were analyzed by Western blotting with a biotinylated anti-BAZF antibody. Beta-actin was detected as a loading control. (F) A molecular schema of BAZF-CBF1 interaction designed by the data in supplemental Figure 3. BAZF-ΔZnF5 is a mutant lacking CBF1-binding capacity. (G) A reporter assay of HEY1 promoter activity. HUVECs were cotransfected with a luciferase reporter plasmid containing human HEY1 promoter, along with the plasmids encoding N1ICD, BAZF, and/or BAZF-ΔZnF5. Luciferase activities are represented as arbitrary units. Error bars show ± SD of 5 samples from 3 independent experiments (***P < .001). NS indicates not significant. (H) A ChIP assay using an anti-CBF1 antibody in HUVECs infected with AdBAZF or AdGFP. The CBF1 binding element on human HEY1 and HEY2 promoters was detected in the immunoprecipitates by quantitative RT-PCR.