Figure S1

A Non-lesional Lesional

GEN001 Non-lesional GEN001 Lesional

Normal Negative controlNegative control

B

C

Figure S1. Dermal Populations of CARD14+ cells are found in human skin. Immunohistochemistry for CARD14 on fixed, frozen skin sections of (A) non-lesional and lesional classical psoriasis, (B) non-lesional and lesional skin from patient GEN001, with a confirmed over-active mutation in CARD14 (Jordan CT et al, Am J Hum Genet, 2012 May 4;90(5):784; PMID22521418), and (C) normal human skin. Lower right image is the negative staining control, performed in classical psoriasis lesional skin. Arrows point to dermal CARD14+ cells. Representative images are shown, bar = 10μm.

Normal

Non-lesional

A

B

Figure S2

CARD14CD11c

CD11c and CARD14

CARD14CD31

CD31 and CARD14 CD3 and CARD14

CD3 CARD14

CD3 and CARD14

CD3 CARD14 CARD14CD11c

CD11c and CARD14

Figure S2. Dermal CARD14 is expressed in endothelial cells in normal and non-lesional skin. Two color immunofluroscence on frozen skin sections of (A) normal and (B) non-lesional skin demonstrates that CARD14 (red) does not colocalize with various cell markers (green); CD3 (T-cells), CD11c (dendritic cells), and CD163 (macrophages), but does colocalize with CD31+ endothelial cells (green) in both normal and non-lesional skin. Representative images shown; bar = 10μm.

CARD14CD163

CD163 and CARD14

CARD14CD163

CD163 and CARD14

CARD14CD31

CD31 and CARD14

A Normal Non-Lesional Lesional

Vimentin and CARD14 Vimentin and CARD14

B

Vimentin and CARD14 and CD31

Lesional

Figure S3

Vimentin

CARD14

CD31

Vimentin VimentinCARD14 CARD14

Vimentin (Fibroblasts/stromal cells)

Vimentin and CARD14

Vimentin CARD14

Figure S3. Dermal CARD14 colocalizes with vimentin+CD31+ cells. (A) Two-color immuno-fluroscence demonstrates CARD14 (red) colocalizes with some vimentin (green) cells in normal, non-lesional, and lesional skin. (B) Triple-color immunofluroscence staining in lesional skin demonstrates that the dermal CARD14+ (red) and vimentin+(green), colocalize with the CD31+ endothelial cells (blue). Representative image shown; bar = 10μm.

Non-lesional aorta Lesional aortaA

B

Figure S4

CD31 and CARD14

CD31 CARD14

Figure S4. CARD14 is also expressed in aortic endothelial cells. (A) Immunohistochemistry for CARD14 on fixed, frozen sections of aorta, obtained from cadavers with athelerosclerotic disease, classified as non-lesional (normal apearing) or lesional (atherlosclerotic) based on macroscopic observation. Negative control shown far right. (B) Two-color immunofluroscence for CD31 (green) and CARD14 (red) in aorta. Representative images are shown; bar = 10μm.

Negative Control

Non-lesional aorta Lesional aorta

CD31 CARD14

CD31 and CARD14

Published Psoriasis Gene Sets Enriched in GEN001 (CARD14 mutation) LS vs NL Transcriptome

Published Psoriasis Gene Sets Enriched in NIH(CARD14 mutation) LS vs post-Rx Transcriptome

A

B

Figure S5

NIH.PS.1

GEN001

WU.PS.10

RU.PS.1

RU.PS.2

Figure S5. Transcriptomic profile of patients with CARD14 muations. (A) Principle Component Analysis (PCA) for the gene expression of normal, LS, and NL or post-treatment biopsies of patients with confirmed CARD14 mutations (GEN001 and NIH.PS.1) and classical psoriasis (RU and WU samples). (B) Gene Set Enrichment Analysis (GSEA) of published psoriasis upregulated and downregulated transcriptomes (DEGs) compared to gene expression profile of patients with confirmed CARD14 mutations. cs = connectivity score. Additional information on bioinfomatics is located in the Material and Methods

WU

.HD

.013

N

WU

.HD

.011

N

Nor

mal

_2

Nor

mal

_1

WU

.PSO

.010

_PN

NL_

9

GEN

001_

PN

NL_

51

Pust

ular

_pos

ttrea

t

WU

.PSO

.010

_PP

Pust

ular

_pre

treat

GEN

001_

PP

LS_1

0

LS_5

2

aIl17effectW2_Psoriasis Down

MAD3 Psoriasis Up

Additive IL17andIL22 KC

KC IL1 Down

RHE IL17 Up

Additive IL17andTNFa KC

KC IL17andTNF Up

Synergistic IL17andIL22 KC

KC IL1 Up

KC TNF notIL17 Down

Synergistic IL17andTNFa KC

KC TNF notIL17 Up

KC IL17 notTNF Up

KC IL17andTNF Down

KC IL17 notTNF Down

RHE IL17 Down

MAD3 Psoriasis Down

aIl17effectW2_Psoriasis Up

−1 0 1Row Z−Score

Color Key

NLGEN001

NLNL NL LSGEN001

LSLS LSNIH

LSNIH

POSTN NN N

Figure S6.

A

B

Atherosclerosis Signaling

LPS/IL-1 Mediated Inhibition of RXR Function

Pathogenesis of Multiple Sclerosis

IL-17A Signalling in Gastric Cells

Role of Hypercytokinemia/hyperchemokinemia in the Pathogenesis of In�uenza

Role of Macrophages, Fibroblasts, and Endothelial Cells in Rheumatoid Arthritis

IL-10 Signalling

Interferon Signalling

Role of IL-17A in Psoriasis

MAD3

NIH (LS-POST)

GEN001 (LS-NL)

Figure S6. Transcriptomic profile of patients with CARD14 mutations. (A) Ingenuity Pathway Analysis (IPA), showing biological pathways significantly upregulated in both classical psoriasis (MAD3) as well as patients with confirmed CARD14 mutations. (B) Heat map of cyotkine GSVA (gene set variation analysis)-derived scores in normal, LS, and NL or post-treatment biopsies of patients with confirmed CARD14 mutations (GEN001 and NIH.PS.1) and classical psoriasis. Additional information on bioinformatics is located in the Materialsand Methods.

IL-8

fl CARD14

sh C

ARD14

G117S

E138A

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e E

xpre

ssio

n to

hA

RP

CCL2

fl CARD14

sh C

ARD14

G117S

E138A

0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e E

xpre

ssio

n to

hA

RP

p = 0.13

CXCL10

fl CARD14

sh C

ARD14

G117S

E138A

0

1

2

3

Rel

ativ

e E

xpre

ssio

n to

hA

RP

CCL5

fl CARD14

sh C

ARD14

G117S

E138A

0

1

2

3

4

5

Rel

ativ

e E

xpre

ssio

n to

hA

RP

CXCL1

fl CARD14

sh C

ARD14

G117S

E138A

0.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e E

xpre

ssio

n to

hA

RP

*

Figure S7

Figure S7. Transfection of psoriasis-associated CARD14 mutations into dermal endothelial cells resulted in increased expression of several chemokines. Quantitative RT-PCR for various chemokines in endothelial cells transfected with wild-type (full-length (fl) and short (sh) CARD14)(white), or psoriasis-associated CARD14 mutation constructs: G117S (gray) and E138A (black). N= 3 per group. Error bars represent the standard error of the mean.

Figure S8

A

B WT CARD14E138A CARD14

CE-selectin

WT

Tx

E138A

Tx

0

2000

4000

6000

8000

10000

MFI

P-selectin

WT Tx

E138A

Tx0

200

400

600

MFI

ICAM

WT T

x

E138A

Tx0

5000

10000

15000

20000

MFI

VCAM

WT

Tx

E138A

Tx

0

5000

10000

15000

20000

25000

MFI

Figure S8. Minimal upregulation of cell adhesion molecule protein expression on E138A transfected HDBECs. (A) qRT-PCR analysis of E-selectin, P-selectin, ICAM, and VCAM on wild-type (fl and shCARD14) transfected HDBECs, or HDBECs transfected with psoriasis associated mutations (G117S and E138A). Expression is presented as relative to the housekeeping gene, hARP. N = 2-3 per group. Error bars represent the standard error of the mean. (B) and (C) Expanded HDBECs transfected with the wild-typeCARD14 or the E138A psoriasis-associated CARD14 mutation (N=1) were stimulated with TNF-alpha overnight and then assessed for cell surface protein expression of adhesion molecules by flow cytometry; (B) histograms and (C) corresponding mean fluroscence intensity (MFI) are shown.

E-selectin

fl CARD14

sh C

ARD14

G117S

E138A

0.00

0.02

0.04

0.06

Rel

ativ

e E

xpre

ssio

n to

hA

RP

P-selectin

fl CARD14

sh C

ARD14

G117S

E138A

0.000

0.002

0.004

0.006

Rel

ativ

e E

xpre

ssio

n to

hA

RP

ICAM

fl CARD14

sh C

ARD14

G117S

E138A

0.00

0.05

0.10

0.15

0.20

Rel

ativ

e E

xpre

ssio

n to

hA

RP

VCAM

fl CARD14

sh C

ARD14

G117S

E138A

0.00

0.01

0.02

0.03

0.04

Rel

ativ

e E

xpre

ssio

n to

hA

RP

Figure S9

IL-8

PBSIL-

17TNFa

IFNg

IL-17

+TNFa

IL-17

+IFNg

0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e E

xpre

ssio

n to

hA

RP

CCL2

PBSIL-

17TNFa

IFNg

IL-17

+TNFa

IL-17

+IFNg

0

1

2

3

Rel

ativ

e E

xpre

ssio

n to

hA

RP

CXCL1

PBSIL-

17TNFa

IFNg

IL-17

+TNFa

IL-17

+IFNg

0.0

0.2

0.4

0.6

0.8

Rel

ativ

e E

xpre

ssio

n to

hA

RP

CCL5

PBSIL-

17TNFa

IFNg

IL-17

+TNFa

IL-17

+IFNg

0.00

0.02

0.04

0.06

0.08

0.10

Rel

ativ

e E

xpre

ssio

n to

hA

RP

CXCL10

PBSIL-

17TNFa

IFNg

IL-17

+TNFa

IL-17

+IFNg

0

2

4

6

8

10

Rel

ativ

e E

xpre

ssio

n to

hA

RP

Figure S9. Psoriatic cytokines upregulate chemokines in dermal blood endothelial cells. Human dermal blood endothelial cells were cultured with either vehicle control (PBS), IL-17 (100ng/ml), TNF-alpha (20ng/ml), IFN-gamma (20ng/ml), or combinatations. RNA was isolated after 12 hours of exposure to cytokines, and expression of various chemokines was determined using qRT-PCR (as described in the materials and methods). Error bars represent the standard error of the mean. Significance was determined by comparison of cytokine treatment versus PBS, * p < 0.05, **p < 0.01 using a paired t-test. N=3.

* *

**

*

*

* *

*

*

Figure S10

Figure S10. Single stained flurochrome slide controls for triple-color immunofluroscence studies. Representative images of each single-flurochrome stained sample using the the green, red, or blue laser are shown at 20X.

Table S1: Antibodies used for immunohistochemistry/immunofluorescence  

 a All antibodies are murine monoclonal unless otherwise stated.

Antigen Manufacturer Clonea Dilution

Amplification/detection (secondary antibodies)

CARD14 Sigma-Aldrish Rabbit polyclonal

1:600 Goat anti-rabbit Ig rhodamine red

CD3 BD SK7 1:100 Goat anti-mouse IgG1 A-488

CD11c BD B-ly6 1:100 Goat anti-mouse IgG1 A-488

CD163-FITC

Acris 5C6-FAT 1:100 Goat anti-mouse IgG FITC

Vimentin Thermo Scientific

Ab2 1:100 Goat anti-mouse IgG1 A-488

CD31 BD WM59 1:100 Goat anti-mouse IgG1 A-488 or A-647

PAL-E abcam ab8853 1:50 Goat anti-mouse IgG1 A-488

LYVE-1 R&D 537028 1:200 Goat anti-mouse IgG1 A-488

pNFkB (ser276)

Santa Cruz Rabbit polyclonal

1:100 Zenon conjugated A-647

CXCL1 Acris Rabbit polyclonal

1:100 Goat anti-rabbit Ig rhodamine red

CCL2 Novus Biologicals

MNA1 1:100 Zenon conjugated A-568

CCL5 abcam VL-1 1:200 Goat anti-mouse IgG2b A-568