Figure 5: Direct activation of KLF4 expression by the Jagged1/Notch signalling.

(a) Western blot to examine the expression levels of Jagged1, BMI1, KLF4 and Twist1 in various stable clones. Actin was used as an internal control. (b) Transient transfection assays were performed using a Jagged1 expression vector with a KLF4 promoter-driven reporter construct harbouring either a wild-type or mutated NICD/CSL-responsive site. The pcDNA3 control-transfected lane was selected as the control group versus the pcDNA3-Jagged1-transfected lane for each reporter construct. (c) ChIP experiments were performed using an IgG or anti-NICD antibody to detect their binding to two regions in the KLF4 gene promoter. The cell clones of OECM1 control (cont.), OECM1-Twist1 and OECM1-Twist1 with Jagged1 knockdown were used. qChIP results of NICD binding to the KLF4 promoter regions were also shown. The OECM1 control clone was selected as a control. The Hes1 promoter was used as a positive control for NICD binding (Supplementary Fig. 28c). Data shown in panels b,c are mean±s.d. of three independent experiments. Asterisk (*) indicates statistical significance (P<0.05 by Student’s t-test) between experimental and control transfections (b) or control clones (c).