Supplementary Fig. 1!

a! b! c!

d! Nogo-A/B-deficient!WT!

eNOS!β-actin!

MLEC!Nogo-B!

Nogo-A/B-deficient !WT!nNOS!

β-actin!

Nogo-B!Aorta!

e!

-9 -8 -7 -6 -5 -40

4080

120160200240280

Diam

eter

(μm

)WTNogo-A/B-deficient

PE (LogM)-12-11-10 -9 -8 -7 -6 -5

04080

120160200240280

Diam

eter

(μm

)

U-46619 (LogM)-8 -7 -6 -5

04080

120160200240280

Diam

eter

(μm

)

S1P (LogM)

0.0

0.5

1.0

1.5

nNOS

/β-a

ctin

(a.u

.)

0.0

0.5

1.0

1.5

eNOS

/β-a

ctin

(a.u

.)

WT Nogo-A/B-deficient

Nature Medicine: doi:10.1038/nm.3934

a! b!

d

microsomes !f!Nogo-B!

HSP90!

LC1!

Calnexin!

WT !Ng-A/B-deficient! Ng-A/B-

deficient !WT !

soluble! fraction !

Supplementary Fig. 2!

c!Control!

myriocin 0.3 nM!

myriocin 1 nM!

myriocin 3 nM!

myriocin 10 nM!

myriocin 30 nM!

e!

0.01 0.1 1 10 1001000

0

30

60

90

120

Dose (nmol/L)

Sphi

ngan

ine

inhi

bitio

n (%

)

d!

0

500

1000

1500

2000

2500To

tal c

eram

ide

(pm

ol*m

g-1*m

l -1)

WT Nogo-A/B-deficient

C16-C

er

C24-C

er

C24:1-Ce

r0

100

200

300

400

pmol*mg-1 *ml-1

C14-C

er

C18-C

er

C18:1-Ce

r

C20-C

er

C20:1-Ce

r

C22-C

er

C22:1-Ce

r

C26-C

er

C26:1-Ce

r

dhC16-Cer

0102030405060

pmol*mg-1 *ml-1 ** **

dh-S1PdhS

ph Sph

S1P

0246850

100

150

pmol*mg-1 *ml-1

0 0.1 0.3 1 3 10 30 100 300 !

HUVEC + myriocin (nM)!

PS!SM!

Sphinganine!

Ceramide!

Nature Medicine: doi:10.1038/nm.3934

Nature Medicine: doi:10.1038/nm.3934

Supplementary Fig. 4 !

a!

d! e! f! g! h!

Nogo-B!

HSP90!

VE-cadherin!

αSMA!

b!

Nogo-B!

HSP90!

VE-cadherin!

αSMA!

c!

α-SMA! Nogo-B!

EC-!

Nog

o-A/

B-de

ficie

nt!

IB4! Nogo-B!

Nog

o-A/

Bf/f!

α-SMA!50 μm!Nogo-B!

SMC

-!N

ogo-

A/B-

defic

ient!

0.0

0.4

0.8

1.2

1.6

Nog

om

RN

A ex

pres

sion

Nogo-A/Bf/f

EC-Nogo-A/B-deficient

0.0

0.4

0.8

1.2

1.6

2.0

αSM

A m

RN

A ex

pres

sion VSMC

EC

0.0

0.4

0.8

1.2

1.6

2.0

VE-c

adhe

rin

mR

NA

expr

essi

on VSMCEC

0.0

0.4

0.8

1.2

1.6

Nog

om

RN

A ex

pres

sion

Nogo-A/Bf/f

SMC-Nogo-A/B-deficient

-9 -8 -7 -6 -5 -40

20

40

60

80

100

% In

itial d

iam

eter

Nogo-A/Bf/f

SMC-NogoA/B-deficientEC-NogoA/B-deficient

Phe (LogM)

Nature Medicine: doi:10.1038/nm.3934

SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. The loss of Nogo-B does not affect the vasoconstriction in response to

pharmacological agents. Concentration-response curves of WT and Nogo-A/B-deficient MA in response to (a)

PE, (n = 8 WT vs. n = 7 Nogo-A/B-deficient), (b) U-46619, (n = 5 per group) and (c) S1P (n = 10 WT vs. n = 6

Nogo-A/B-deficient). WB analysis and relative quantification of (d) eNOS on MLEC from WT and Nogo-A/B-

deficient mice, n = 4 isolations per group, and (e) nNOS on WT and Nogo-A/B-deficient aortas (n = 4 aortas

from 4 mice per group). β-actin was used as loading control. Data were expressed as the mean ± s.e.m.

**P<0.01 compared to WT group. Statistical significance was determined by two-way ANOVA followed by

Bonferroni’s post-test (a–c) and unpaired t-test (d–e).

Supplementary Figure 2. Sphingolipids levels in vascular SMC and SPT assay in EC. WT and SMC-Nogo-

A/B-deficient (a) total ceramide levels and (b) individual sphingolipid species measured by LC/MS as described

in Methods. n = 4 independent VSMC isolation/group; five mice were used for each VSMC isolation. Mean ±

s.e.m. (c) Pharmacological inhibition of SPT activity by increasing concentrations of myriocin in HUVEC. The

inhibition was assessed using [3H]serine as substrate in endothelial cell lysates as described in Methods.

Ceramide-C18 (ceramide), sphinganine, phosphatidylserine (PS) and sphingomyelin-C18:1 (SM). (d) Extracted

lipids were separated by TLC and [3H]sphinganine was detected by using TLC scanner (TLC analyzer RITA,

Raytest Straubenhardt) and reading values were quantified using a standard curve of [3H]serine.

(e) Inhibition of SPT activity, expressed as percent of the control, by increasing concentrations of myriocin. (f) WB analysis for Nogo-B, SPTLC1, Calnexin and HSP90 in microsomes isolated from WT and Nogo-A/B-

deficient mouse lung. Unpaired t-test was performed to evaluate the statistical significance.

Supplementary Figure 3. Effects of W146 and myriocin on vascular tone regulation. (a) Ach cumulative

concentration-response curve in WT mesenteric arteries treated with myriocin (0.3 mg/Kg i.p.) or vehicle in

presence of L-NAME, (left, n = 7 per group), indomethacin, (middle, n = 13 vehicle vs. n = 9 myriocin), and

their combination, (right, n = 5 vehicle vs. n = 6 myriocin). (b) Concentration-response curve of S1P in MA from

WT and Nogo-A/B-deficient mice incubated with L-NAME or vehicle. n = 4 per group. (c) WT mesenteric

arteries treated with W146 (100 nM, 45 min) or vehicle, were incubated with L-NAME and the increase in the

basal tone was measured as internal diameter decrease over the baseline. (n = 11 ctrl vs. n = 5 W146 100

nM). (d) The active diameter of WT mesenteric arteries treated with different concentrations of W146 or vehicle

in response to stepwise increase in intraluminal pressure. (n = 12 ctrl vs. n = 5 W146 100 nM and 1 μM). (e)

Vasoconstriction induced by PE following W146 treatment of WT mesenteric arteries. (n = 8 ctrl vs. n = 9 W146

100 nM). (f) Ach-induced vasodilation in WT mesenteric arteries following W146 (100 nM) treatment. Given

that basal and PE-stimulated tone is different after W146 treatment, the following data are expressed as

Nature Medicine: doi:10.1038/nm.3934

absolute internal diameter (μm) of MA. (n = 21 ctrl vs. n = 7 W146 100 nM). (g) In another set of experiments,

after incubation with W146, Ach cumulative concentration-response curve was performed in presence of: L-

NAME (left, (n = 8 ctrl vs. n = 5 W146 100 nM),.indomethacin (middle, n = 7 per group) and their combination

(right, n = 6 per group). (h) Schematic summary of myriocin and W146 effects on the vascular tone. Data are

expressed as the mean±s.e.m. *P<0.05; **P<0.01 compared to vehicle. Statistical significance was determined

by two-way ANOVA followed by Bonferroni’s post-test (a–b, d–g) and unpaired t-test (c).

Supplementary Figure 4: Characterization of the Cre-mediated excision of Nogo-B in EC and VSMC of EC-

Nogo-A/B-deficient and SMC-Nogo-A/B-deficient mice. (a) IF staining of MA cross-sections for: (top panels)

Nogo-B (red) and α-smooth muscle actin (αSMA; green) in NogoA/Bf/f MA; (central panels) Nogo-B (red) and

EC with isolectin-B4 (IB4, green) in SMC-Nogo-A/B-deficient MA; and (bottom panels) Nogo-B (red) and

αSMA (green) in SMC-Nogo-A/B-deficient MA. (b) Western blot analysis for Nogo-B in EC isolated from

NogoA/Bf/f and EC-Nogo-A/B-deficient lungs. (c) Western blot analysis for Nogo-B in VSMC isolated from

NogoA/Bf/f and SMC-Nogo-A/B-deficient thoracic aortas as described in Methods. Heat shock protein 90

(HSP90) was used as loading control. Vascular endothelial cadherin (VE-cadherin) and αSMA were used as

lineage markers of EC and smooth muscle cells/fibroblasts, respectively. (d) RT-PCR for Nogo-B in EC

isolated from NogoA/Bf/f and EC-Nogo-A/B-deficient lungs; RT-PCR for (e) αSMA and (f) VE-cadherin in

endothelial and smooth muscle cells mRNA isolated from NogoA/Bf/f and SMC-Nogo-A/B-deficient thoracic

aortas as described in Methods. (g) Nogo-B expression of thoracic aorta VSMC mRNA isolated from

NogoA/Bf/f and SMC-Nogo-A/B-deficient mice (h) PE-concentration-response curves of Nogo-A/Bf/f, EC-Nogo-

A/B-deficient and SMC-Nogo-A/B-deficient MA.

Nature Medicine: doi:10.1038/nm.3934