Cellular Effect of CADASIL Mutants of NOTCH3 Figure 5. Clearance of mutant NOTCH3 proteins. 293 cells (n = 3 replicate wells per group) were transiently transfected with NOTCH3-luciferase constructs and split into identical 24 well chambers after 24 hours. After another 24 hours incubation, cells were treated with cycloheximide (100 ug/ ml) in serum free media to arrest all protein synthesis, and groups of cells were analyzed at time points shown, when cell lysates were prepared and assayed for luciferase enzyme content. (A) The levels of NOTCH3-luciferase activity at the beginning of the time course did not differ significantly between any of the wildtype or mutant combinations. (B) Wildtype NOTCH3 ectodomain was more rapidly cleared than mutant NOTCH3 ectodomains. When cotransfections were performed with wildtype and mutant NOTCH3, proteins were cleared at an intermediate speed. (C) Quantification of a single time point (3 hours) is shown in a bar graph format to facilitate interpretation. The wild type protein is cleared most rapidly. Both CADASIL mutations slow clearance rate significantly. When mutants and wildtype protein were expressed together, there was an intermediate level of stability for R90C/wildtype protein. The C49Y/wildtype mixture was degraded at a similar rate as C49Y protein. Data is representative of three independent experiments. (*Significant compared to wildtype protein, #significant compared to all other groups; p,0.05.) Error bars represent standard deviations. doi:10.1371/journal.pone.0044964.g005 creases in transcript levels in cocultures (rat smooth muscle mRNA was determined using species specific primers and qRT-PCR; Figure 6). We did not detect a consistent difference between the inhibitory effects of transfected full-length WT or mutant NOTCH3. Notch signaling activates gene expression by derepressing CBF- 1 responsive elements within promoter. To determine whether decreased smooth muscle mRNA levels could be due to decreased Notch activity, we examined the effects of full-length NOTCH3 overexpression on a prototypical CBF-1 regulated promoter by cotransfecting the HES-luciferase reporter into cells that were then cocultured with ligand producing cells. In transfected H460 cells (Figure 7A), which endogenously express only NOTCH3, cotransfection with wildtype or mutant full-length NOTCH3 A7R5 cells cocultured with L( ), Jagged1( ) or Delta1 cells( ) Figure 6. Inhibition of Notch-mediated smooth muscle marker expression by NOTCH3 transfection. A7R5 cells (n = 3 replicate wells per group) were cotransfected with NOTCH3 constructs shown. Control was empty vector pCMV-Sport6. After coculture with ligand expressing cells, mRNA levels of genes indicated were measured by quantitative RT-PCR using primers that detect rat sequences present in rat A7R5 cells but not mouse genes. Analysis was performed as noted in the methods and the display show the average level of gene induction in three independent experiments. * indicates differences between NOTCH3 transfected cells versus control cells and # denotes differences between mutant NOTCH3 and wildtype NOTCH3 transfectants (p,0.05). Error bars represent standard deviations. doi:10.1371/journal.pone.0044964.g006 PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44964