Supplemental Figure 1. Proliferating and involutingIH morphology. Representative H&E staining ofproliferating (n=3) and involuting IHs (n=3). Boxed areaenlarged to right. Black arrowheads mark endothelialcells. Blue arrowheads mark perivascular cells. Blackarrows mark red blood cells. Scale bars - 100μm. IH,infantile hemangioma.

Prol

ifera

ting

Invo

lutin

gSupplemental Figure 1, Edwards et al.

Supplemental Figure 2, Edwards et al.

A αSMA MergedGLUT1

Prol

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CD31 MergedGLUT1

NOTCH3 CD31 Merged

Invo

lutin

g

Supplemental Figure 2. High magnification images of proliferating and involuting IHspresented in Figure 1. A) GLUT1 and CD31 co-staining. White arrowheads markGLUT1+/CD31+ cells. Yellow arrowheads mark GLUT1+/CD31- cells. 3, Proliferating IHn=2, involuting IH n=3. B) GLUT1 and αSMA co-staining. Yellow arrowheads markαSMA+/GLUT1- perivascular cells. Proliferating IH n=2, involuting IH n=7 C) NOTCH3 andCD31 co-staining. White arrowheads mark NOTCH3+/CD31+ cells. Yellow arrowheadsmark NOTCH3+/CD31- cells. Proliferating IH n=4, involuting IH n=5. D) NOTCH3 and αSMAco-staining. White arrowheads mark NOTCH3+/αSMA+ perivascular cells. Yellowarrowheads mark NOTCH3+/αSMA- lumenal cells. Proliferating IH n=5, involuting IH n=8.Scale bars - 25μm. Total number of IH specimens assessed for each antigen presented inSupplemental Table 3. αSMA, alpha smooth muscle actin; GLUT1, glucose transporter 1;IH, infantile hemangioma.

B

Supplemental Figure 3. NOTCH3 is expressed in asubset of Glut1+ IH cells. Proliferating and involuting IHspecimens stained for NOTCH3 and GLUT1. Whitearrowheads mark NOTCH3+/GLUT1+ cells. Yellowarrowheads mark GLUT1+/NOTCH3- lumenal cells.Proliferating IH n=4, involuting IH n=2 Scale bars - 50μm.GLUT1, glucose transporter 1; IH, infantile hemangioma.

Supplemental Figure 3, Edwards et al.

Prol

ifera

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Notch3 Glut1 Merged

Supplemental Figure 4, Edwards et al.

D CD31 MergedNG2

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C αSMA MergedNG2

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Supplemental Figure 4. High magnification images of proliferating and involuting IHspresented in Figure 2. A) PDGFRβ and αSMA co-staining. White arrowheads markPDGFRβ+/αSMA+ perivascular cells. Yellow arrowheads mark PDGFRβ+/αSMA- cells.Proliferating IH n=2, involuting IH n=3. B) PDGFRβ and CD31 co-staining. White arrowheadsmark PDGFRβ+/CD31+ cells. Yellow arrowheads mark PDGFRβ+/CD31- perivascular cells.Proliferating IH n=3, involuting IH n=2. C) NG2 and αSMA co-staining. White arrowheads markNG2+/αSMA+ cells, and yellow arrowheads mark NG2+/αSMA- cells. Proliferating IH n=7,involuting IH n=7. D) NG2 and CD31 co-staining. White arrowheads mark NG2+/CD31+ cells,and yellow arrowheads mark NG2+/CD31- cells. Proliferating IH n=7, involuting IH n=3. Scalebars - 25μm. Total number of IH specimens assessed for each antigen presented inSupplemental Table 3. αSMA, alpha smooth muscle actin; GLUT1, glucose transporter 1; IH,infantile hemangioma; NG2, neuron-glial antigen 2.

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Supplemental Figure 5. Expression of endothelial and perivascular markers inisolated HemSCs. A) CD133+ HemSCs (n=3, done in duplicate) stained for NOTCH3,NG2, GLUT1, PDGFRβ, αSMA, and CD31. Scale bars - 50μm. B) NG2, GLUT1 andCD31 (red line) and control IgG (blue line) FACS of HemSCs (n=3, done in duplicate).C) Representative data of HemSC NOTCH3 and PDGFRB transcript levels determinedby qRT-PCR and normalized to BACTIN. (n=3, done in duplicate) Error bars represent ±S.D. αSMA, alpha smooth muscle actin; GLUT1, glucose transporter 1; IH, infantilehemangioma; HemSC, hemangioma stem cell; NG2, neuron-glial antigen 2.

A

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Supplemental Figure 5, Edwards et al.

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Supplemental Figure 6. Characterization of NOTCH3knockdown HemSCs. A) Relative NOTCH1, HEY1 and HEY2transcript levels. Representative data from 4 independent N3KDHemSC populations and matching Scr HemSC populationsdetermined by qRT-PCR and normalized to BACTIN. Error barsrepresent ± S.D. n.s. - not significant. Student T-Test. B) RelativeVEGFA, ANG1, and ANG2 transcript levels. Representative datafrom 2 independent N3KD HemSC populations and matching ScrHemSC populations determined by qRT-PCR and normalized toBACTIN. Error bars represent ± S.D. *p<0.01, **p<0.005. Student T-Test. ANG1, angiopoietin 1; ANG2, angiopoietin 2; HemSC,hemangioma stem cell; N3KD, Notch3 knockdown,

Supplemental Figure 6, Edwards et al.

A

BVEGFA ANG1 ANG2

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Supplemental Figure 7, Edwards et al.

ScrH

emSC

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differentiation media

Supplemental Figure 7. NOTCH3 knockdownblocks HemSC mural cell differentiation.Scrambled HemSCs (Scr HemSC) or NOTCH3knockdown HemSCs (N3KD HemSC) weregrown in growth or mural cell differentiationmedia for 2 weeks and stained for αSMA. Scalebars - 50μm. (n=3 HemSC populations done induplicate) αSMA, alpha smooth muscle actin;HemSC, hemangioma stem cell.

αSMA

αSMA αSMA

αSMA

Matrigel ECFC HemSC HemSC+ECFC

Supplemental Figure 8. HemSC/ECFC xenograftsrobustly develop an IH like phenotype. HemSCs,ECFCs or both ECFCs and HemSCs in 1:1 ratio wereresuspended in Matrigel, and subcutaneouslyimplanted into the flanks of immunocompromisedmice. Matrigel without cells served as a control.Xenografts were evaluated at Day 14 post-implantation. Two independent experiments were doneand representative data presented. Top) Grossappearance of explanted Matrigel. Bottom) H&Estaining of representative xenograft sections.Arrowheads mark large caliber red-blood cellcontaining IH-like vessel. Scale bar - 50μm. IH,infantile hemangioma; HemSCs, hemangioma stemcells; ECFCs, endothelial colony forming cells.

Supplemental Figure 8, Edwards et al.

Supplemental Figure 9. NOTCH3 knockdown in HemSCs inhibits IH development ina HemSC-only xenograft mouse model. A) Quantification of vessel density and caliber.Results representative of n = 3 HemSC populations (2 implants each). Error barsrepresent ± S.D. n.s., not significant, *p<0.0001. Student T-Test. B) αSMA+ mural celldensity determined as mean mural cell αSMA signal intensity normalized to IH endothelialGLUT1 signal intensity. Average mural cell αSMA expression determined as mean αSMAsignal intensity normalized DAPI+/αSMA+ cell number. Results representative of n = 3HemSC populations (2 implants each). Error bars represent ± S.D. n.s., not significant,**p<0.01. Student T-Test. αSMA, alpha smooth muscle actin; GLUT1, glucose transporter1; HemSC, hemangioma stem cell; MC, mural cell.

Supplemental Figure 9, Edwards et al.

n.s.

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Supplemental Figure 10. The NOTCH3 inhibitor,human NOTCH3 Decoy (N3 Decoy), inhibits JAG1activation of Notch signaling. A) N3 Decoy encodes thesignal peptide (SP) and EGF-like repeats 1-24 of humanNOTCH3 fused to in frame with human IgG FC. B)Notch/CSL signal activation measured in HeLa cellsexpressing full-length rat NOTCH1, N3 Decoy and aNotch luciferase reporter (11CSL-Luc) co-cultured withHeLa cells expressing the Notch ligand, JAG1. Addition ofCompound E, a gamma-secretase inhibitor (GSI), wasused as a control and inhibited Notch activation of the11CSL-Luc reporter. Co-culture assays were performed intriplicate and repeated three times. Data presented asmean luciferase fold induction ± S.D. *p< 0.02, **p<0.005,Student T-Test. CSL, C promoter binding factor1/Suppressor of Hairless/Lag-1.

Supplemental Figure 10, Edwards et al.

A

FC Control NOTCH3 decoy

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US Doppler US Doppler

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Supplemental Figure 11. Design and validation of the NOTCH3 DecoyHemSC/ECFC xenograft study. A) Schematic of experimental time-line. B)Staining with antibodies against human FC of livers (Day 21) from mice injected anadenovirus encoding either N3 Decoy or FC. Liver sections from un-injected miceserved as a negative control. Scale bar - 50μm. C) Western blot with anti-FCantibody of sera (Day 21) collected from mice injected with the adenovirusencoding FC (n=4). D) Western blot of sera immunoprecipitated with antibodiesagainst human FC and then probed with anti-FC antibody (n=5). N3 Decoy was notdetected in the sera of mouse 4 and analysis of this xenograft was excluded fromthe study. ECFC, endothelial colony forming cell; HemSC, hemangioma stem cell;N3 Decoy; NOTCH3 Decoy

Supplemental Figure 11, Edwards et al.

CONTROL DECOY0

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Supplemental Figure 12, Edwards et al.

αSMA αSMAFC NOTCH3 decoy

Supplemental Figure 12. NOTCH3 Decoy treatment has no effects on αSMA+perivasculature expression in hepatic vasculature. A) Liver sections from micetreated with FC (control) or NOTCH3 Decoy (n=2 populations; n=4 xenografts each)stained for αSMA. B) Mean αSMA expression determined as mean αSMA signalintensity normalized DAPI+/αSMA+ cell number. (n=2 populations; n=4 xenograftseach) n.s., not significant. Student T-Test. αSMA, alpha smooth muscle actin.

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