Fig. 4. Notch modulates Wnt pathway transcriptional activity in both Drosophila and vertebrate cells. (A) Diagram of the Notch molecules used. (B) Ectopic activation of the Wnt signalling pathway was observed in Drosophila clone8 (cl8) cells in the presence of Notch dsRNA (104-fold activation compared to no dsRNA), but not GFP dsRNA (1.1-fold activation). (C,D) In Drosophila SL2 (C), or S2R⁺ cells (D) Wnt signalling was induced with an oncogenic form of ß-catenin, S37A ß-catenin (Schweizer and Varmus, 2003), the presence of a membrane tethered form of Notch (TNotch) significantly reduced the level of ectopic Wnt signalling (C,D). (E-H) N-N1 (delN-N1) cleaves spontaneously to release the NICD domain of Notch1 as shown by the strong activation of the CBF1 reporter (H), whereas LNR-N1 rarely cleaves as shown by the weak activation of the CBF1 reporter (H) (Mumm et al., 2000). A further inhibitor of Wnt signalling ExFz8 acts by titrating Wnt (Brennan et al., 2004). Ectopic Wnt signalling was induced with Wnt1, delN-LRP6, Dishevelled, activated ß-catenin or LEF1-VP16 in HEK-293T cells. Both forms of Notch are capable of significantly repressing ectopic Wnt signalling induced by Wnt1, Dsh, and activated ß-catenin (E,F), LNR-N1 effects extended to ectopic Wnt signalling induced by delN-LRP6 and LEF1-VP16. Whereas, ExFz8 repressed ectopic Wnt signalling induced by Wnt1, some small effects on the intracellular mediators of Wnt signalling were observed, such effects have previously been reported (Suzuki et al., 2004) (G).