Figure 3. Inhibition of Twist by Prox1 is required for the upregulation of p53. (A) SK-HEP-1 cells were trasnfected with pcDNA or Prox1 expression vector. After 48 h, ChIP assay was performed by using anti-Prox1 or anti-Twist antibody to detect the binding of these two proteins to human p53 proximal promoter region. (B) Effect of Prox1 on Twist expression was investigated by western blotting. (C) SK-HEP-1 cells were co-transfected human p53 promoter with luciferase (Luc) or Twist shRNA for 48 h. Protein levels of Twist and p53 (upper panel) and luciferase activity (bottom panel) were determined. Promoter activity of cells co-transfected with luciferase shRNA was defined as 100%. *p < 0.05 when compared with the control group. (D) SK-HEP-1 cells were transfected with pcDNA (Con), Prox1 or Prox+Myc-tag Twist vector. After 48 h, RT-PCR and western blotting were performed to study Prox1, Twist and p53 expression.