Figure 4
From: Inhibition of Wnt signalling by Notch via two distinct mechanisms
The inhibition of β-catenin-driven transcription by NICD does not require transcription of its downstream target genes. (A) Schematic showing the structure of the NICD, NICD W1758A and NICD R1994A constructs. (B) Introducing the W1758A mutation into NICD abolishes its ability to induce transcription of the Notch-dependent reporter gene p10xRbpj-luc. In contrast, the R1994A has little effect on NICD function. (C) Both NICD W1758A and NICD R1994A were able to reduce S45Fβ-catenin-driven transcriptional activity. (D) The repressor function of Hes5 is blocked by DNHes5. Expressing Hes5 with VP16-RBPj reduces the ability of VP16-RBPj to activate the pHes1-luc reporter plasmid. This inhibition of VP16-RBPj driven transcription was attenuated, as expected, by expressing a dominant negative form of Hes5. (E) DNHes5 is not able to eliminate the ability of NICD to inhibit S45Fβ-catenin-driven transcriptional activity. (F) siRNA silencing RBPj attenuates the ability of NICD to induce Notch signalling. (G) Inhibition of RBPj by siRNA blocked the ability of NICD to inhibit S45Fβ-catenin-driven transcriptional activity. HEK293T cells were transfected with p10xRbpj-luc or pHes1-luc (B,D,F) to monitor Notch signalling and pTCFAdTATA (C,E,G) to monitor Wnt signalling. Notch signalling was activated by expressing NICD (B,F) or VP16-RBPj (D). Wnt signalling was activated by expressing S45Fβ-catenin (C,E,G). Experiments were performed in triplicate. pRL-CMV was used as a transfection control and cells were lysed 48 h post transfection to determine luciferase activity. Data are presented as mean fold change (± SEM) in RLU (NS P > 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001 one-way ANOVA and Tukey’s post-hoc test, N = 3).
