Cellular Effect of CADASIL Mutants of NOTCH3 Figure 7. Inhibition of Notch mediated transcription by overexpression of full-length NOTCH3. Notch expressing cell lines H460 (A) or A7R5 (B–E) were cotranfected with luciferase reporters and either vector or full length NOTCH3 expression plasmids (n = 3 replicate wells per group). In panels A and B, we show effects of NOTCH3, mutants R90C and C49Y, and control vector on ligand stimulation of HES-luciferase. The effects of cotransfection of NOTCH3 on smooth muscle actin-luciferase (C), smooth muscle MHC-luciferase (D), SM22-luciferase (E, left group) and mutant SM22-luciferase (E, right group) are shown in the lower panels. After one day, cells were overlayed with L, Jagged1, or Delta1 expressing fibroblasts to assess the effect of Notch ligand stimulation. Luciferase assays were performed after 24 hours to quantify the level of Notch signaling. All ligand PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44964