Table 1. Mouse strains used in the study.

Nomenclature	Description	Purpose
TgNotch3^R169C	Mice overexpressing a rat Notch3 protein with the R169C mutation (line 88), 4 fold over the endogenous Notch3 ⁽¹¹⁾	Characterize mutant Notch3-induced structural changes (Figures 1 and S1) and assess Notch3 activity in cerebral arteries (Figure 2)
TgNotch3^WT	Mice overexpressing a rat Notch3 protein with the wild type sequence (line 129), about 4 fold over the endogenous Notch3 ⁽¹¹⁾
non-Tg	Non transgenic littermates

TgNotch3^R169C;Timp3^+/+	TgNotch3^R169C mice with normal expression of TIMP3 ⁽¹⁵⁾	Analyze the involvement of excess TIMP3 in structural changes caused by the R169C mutation (Figure S2)
TgNotch3^R169C;Timp3^+/-	TgNotch3^R169C mice with reduced expression of TIMP3 ⁽¹⁵⁾
Non-Tg;Timp3^+/+	Non transgenic mice with normal expression of TIMP3 ⁽¹⁵⁾
Non-Tg;Timp3^+/-	Non transgenic mice with reduced expression of TIMP3 ⁽¹⁵⁾
TgBAC-TIMP3	Mice overexpressing TIMP3 ⁽¹⁵⁾

Notch3^R170C/R170C	Mice with targeted insertion of the R169C mutation into the endogenous Notch3 locus ¹⁰	Assess the consequence of the presence of the R169C mutation in the endogenous Notch3 locus on the cerebral arteries (Figure 3)
Notch3^WT/WT	Littermate mice with the wildtype Notch3 sequence ¹⁰

TgN3^R169C;Rbpj^del-SMC	Tamoxifen-treated TgNotch3^R169C; SMMHC-Cre^ERT2;Rbpj^flox/flox mice to generate TgNotch3^R169C mice with SMC-specific deletion of Rbpj	Examine the contribution of elevated Notch3 activity to structural changes caused by the R169C mutation (Figure 4)
TgN3^R169C;Rbpj^WT	Tamoxifen-treated TgNotch3^R169C; SMMHC-Cre^ERT2;Rbpj^+/+ mice to generate TgNotch3^R169C mice with wild-type expression of RBPJ
non-Tg;Rbpj^del-SMC	Tamoxifen-treated non-Tg;SMMHC-Cre^ERT2;Rbpj^flox/flox mice to generate non transgenic mice with SMC-specific deletion of Rbpj

non-Tg;Rbpj^WT	Tamoxifen-treated non-Tg;SMMHC-Cre^ERT2;Rbpj^+/+ mice to generate non transgenic mice with wild-type expression of RBPJ

TgNotch3ΔE(B)^Act-SMC	Tamoxifen-treated SMMHC-Cre^ERT2; TgNotch3ΔE from line B to generate mice expressing a constitutively activated Notch3 receptor in SMC	Investigate the consequence of elevated Notch3 activity on cerebral arteries (Figure 5, Figures S3-6)
TgNotch3ΔE(C)^Act-SMC	Tamoxifen-treated SMMHC-Cre^ERT2; TgNotch3ΔE from line C to generate mice expressing a constitutively activated Notch3 receptor in SMC
WT	Tamoxifen-treated SMMHC-Cre^ERT2; non transgenic littermates from lines B and C were used as controls