Notch signalling pathway activity in urothelial cells. Notch activity was determined by the ratio of the Notch-dependent wildtype plasmid pJH26A to the Notch-independent mutant plasmid pJH28A. p850-luc and pGL3 plasmids were used as controls. (A) The mammary carcinoma cell line T47D showed a ratio 11:1 suggesting Notch activity. (B) Results of reporter gene assays in primary urothelial cell cultures (UP) and urothelial cancer cell lines in comparison to T47D. (C-D) Induction of Notch activity after transfection of pIRES2-N1ICD in (C) two normal urothelial cell cultures (UP234 and UP188) or (D) UC cell lines BFTC905, VM-Cub1 and 5637. In contrast to the UP cultures, in UC lines Notch activity decreased upon further increasing plasmid concentration (light grey BFTC905, dark grey VM-Cub1, black 5637). (E) Successful overexpression of N1ICD and NOTCH1 full-length (N1-FL) plasmids after 24 hours in UC lines proven by Western Blot with a Notch antibody detecting the C-terminal fragment (Epitomics) with α-Tubulin as a loading control (F) inducing Notch activity in reporter assays. pJH26A to pJH28A ratios are normalized to the empty vector pIRES2. (G) Reporter gene assay with a HES1 promoter with intact or without CBF1 binding sites driving luciferase expression in the four UC lines. Basal luciferase activities were not significantly different between the reporters, whereas co-transfection of the N1ICD fragment significantly activated the HES1 promoter. (H) Assay for DLL1-dependent activation of the Notch signalling pathway in VM-Cub1 and BFTC905 cells. Notch pathway activity was measured by reporter plasmids cotransfected with or without full-length NOTCH1 (N1-FL) and exposed to immobilized extracellular DLL1 domain or IgG. Changes in the pJH26A/pJH28A reporter ratio were not significant between IgG vs. DLL1 exposure (independent of N1-FL), whereas induction by N1-FL was highly significant (T-tests p < 0.01) in each case.