signaling assay, this could not be experimentally discerned. Indeed, recent studies of purified NOTCH3 ectodomains have demonstrated that mutant (but not WT) NOTCH3 proteins spontaneously form very large aggregates [37]. It is thus likely, since inhibition was seen with transfection of cDNA encoding the ectodomain (Figure S3) or application of purified NOTCH3 ectodomain (Figure 8) that the inhibitory effects we observe are due to competitive inhibition through ectodomain interactions. Lastly, because the activation of the NOTCH pathway by transfection of NICD from NOTCH3 was not affected by NOTCH3 overexpression, inhibition of Notch signaling most likely requires ectodomain interactions. Thus, these cell studies may be consistent the following sequence of events in CADASIL: 1) mutations in NOTCH3 lead to decreased solubility; 2) NOTCH3 accumulates; 3) NOTCH3 accumulation leads to selective impairment of Notch signaling and gene activation. Since multiple Notch proteins are expressed in A7R5 cells, these studies also demonstrate that high levels of NOTCH3 likely inhibit multiple Notch isoforms. Dominant negative effects of mutant NOTCH3 have been tested in only one other in vitro study, which failed to find suppressive effects of the R90C and C428S NOTCH3 mutants in cultured cells [8]. However, the C428S mutation, which causes impaired interactions with ligands, was found later to have partial dominant negative function in mice [61]. It is possible that our observations of signal inhibition were of greater magnitude because of differences in reagents and an experimental paradigm which favors detection of cross inhibitory complexes (longer expression period and avoidance of trypsin with EDTA). Another potential explanation for the differences in our results is that overexpression systems were used in these studies which enable very high levels of expression. Finally, our results differ in that added NOTCH3 was used to assess function of endogenous NOTCH proteins expressed at physiological levels in cells. Previous investigators also did not detect a dominant negative effect of mutant Notch in the R90C transgenic mouse model [60]. But, it is difficult to compare this study to ours, since arteries of these mice likely did not express levels of Notch3 that we were able to achieve in vitro. In addition, our studies using multiple independent smooth muscle promoter luciferase reporters offer a new opportunity to examine relevant smooth muscle targets with improved quantitation compared to in vivo measurements in mice expressing a chromogenic reporter, which could miss modest changes in Notch activity. In sum, the available data suggests that high levels of Notch3 expression in smooth muscle cells potently blocks Notch signaling. The consequences of global Notch inhibition in smooth muscle cells could be functionally significant, since marked arterial changes in mice with global smooth muscle Notch inhibition have been noted by Proweller [63]. These dominant negative effects could be clinically important, since Monet-Lepretre et al. have demonstrated that hypomorphic NOTCH3 mutations that lead to loss of NOTCH function result in more severe cerebral white matter changes on MRI in CADASIL patients [61]. References 1. Tournier-Lasserve E, Joutel A, Melki J, Weissenbach J, Lathrop GM, et al. (1993) Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy maps to chromosome 19q12. Nat Genet 3: 256–259. 2. Chabriat H, Tournier-Lasserve E, Vahedi K, Leys D, Joutel A, et al. (1995) Autosomal dominant migraine with MRI white-matter abnormalities mapping to the CADASIL locus. Neurology 45: 1086–1091. 3. Dichgans M, Mayer M, Uttner I, Bruning R, Muller-Hocker J, et al. (1998) The phenotypic spectrum of CADASIL: clinical findings in 102 cases. Ann Neurol 44: 731–739. Cellular Effect of CADASIL Mutants of NOTCH3 Supporting Information Figure S1 Control experiments that demonstrate negative and positive interactions between NOTCH3 and fragments of the protein TSP2. We show two examples of fragments of TSP2 cotransfected with Notch3 and immunoprecipitated. (A) In the first pairwise interaction test, NOTCH3-HA and TSP2-myc tagged proteins were produced in abundance, but did not coprecipitate. (B) In the second pairwise interaction, another domain of TSP2 was shown to interact with Notch3. These data also demonstrate that HA and myc tags do not mediate protein- protein interactions. (EPS) Figure S2 Full-length NOTCH3 expression does not inhibit NICD-driven HES-luciferase activity. H460 cells were co- transfected with (a) full-length NOTCH3 (or vector), (b) NICD3 (or vector), and (c) HES-luciferase and Renilla-luciferase. After one day, HES-luciferase activity from cells (normalized to Renilla activity) was measured. Basal reporter levels were inhibited by all three full-length NOTCH3 constructs. Constitutive activation of the reporter by NICD was not affected by WT or mutant NOTCH3. This suggests that overexpressed NOTCH3 reduces activation of NOTCH3 signaling through ectodomain interac- tions, since NICD is missing the NOTCH3 ectodomain. * represents significant differences compared to N3-ICD groups. # represents significant differences between vector and NOTCH3 expressing cells (p,0.05; without N3-ICD cotransfection). Error bars represent standard deviations. (EPS) Figure S3 Inhibition of Notch mediated transcription by overexpression of NOTCH3 ectodomain A7R5 cells were cotranfected with HES luciferase reporter and either vector or NOTCH3 ectodomain expression plasmids. After one day, cells were cultured with L, Jagged1, or Delta1 expressing fibroblasts to assess activity resulting from Notch ligand stimulation. Luciferase assays were performed after an additional 24 hours and were normalized to co-transfected Renilla luciferase activity. All ligand- stimulated ectodomain groups exhibited significant inhibition of luciferase activity relative to matched control transfected cells (* p,0.05). Error bars represent standard deviations. (EPS) Acknowledgments We thank Dr. Jimo Borjigin for critique and critical reagents for this study. Author Contributions Conceived and designed the experiments: HM XZ SJL MMW. Performed the experiments: HM XZ GGY SJL. Analyzed the data: HM XZ MMW. Contributed reagents/materials/analysis tools: YEC IP. Wrote the paper: MMW. 4. Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, et al. (1996) Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 383: 707–710. 5. Artavanis-Tsakonas S RM, Lake RJ. (1999) Notch signaling: cell fate control and signal integration in development. Science 284: 770–776. 6. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, et al. (2000) A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5: 207–216. PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e44964