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Supplementary Material: 12 supplementary figures

LRRK2 is a regulator of NFAT that modulates the severity of

inflammatory bowel disease.

Zhihua Liu1, Jinwoo Lee1, Scott Krummey1, Wei Lu1, Huaibin Cai2, Michael J. Lenardo1, *

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 1

Supplementary Fig. 1. Lrrk2−/− mice have normal colonic morphology. Representative

images of hematoxylin and eosin (H&E) staining of colonic sections from unmanipulated

wild type (WT) and Lrrk2−/− mice. Scale bar, 100 µm. Data are representative of three

independent experiments.

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 2

Supplementary Fig. 2. Expression of LRRK2. (a) Immunoblot analysis of LRRK2 and

hsp90 in bone marrow derived macrophages (BMDM) from wild-type or LRRK2−/− mice.

(b) Quantitative real-time PCR analysis (mean ± SEM of triplicates) of LRRK2 in

BMDM, BMDC and CD4+ T cells from wild-type cells, presented as the percent to that in

BMDM cells. Actin was used for total mRNA normalization. Data are representative of

two independent experiments.

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 3

Supplementary Fig. 3. LRRK2 deficiency exacerbates DSS colitis in mice. Mean body

weight as a percent of starting weight (a) and mean clinical scores (b) of wild-type (blue,

n=5) and LRRK2−/− (red, n=5) mice with 6 day treatment of 5% DSS followed by

regularu drinking water. * P < 0.05. ** P < 0.02. Data are representative of two

independent experiments (mean and SEM).

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 4

Supplementary Fig. 4 LRRK2 deficiency exacerbates DSS-induced colitis in a cell-

intrinsic manner. Left, mean body weight as a percent of starting weight and right, mean

clinical scores of wild-type (n=5) and Lrrk2−/− (n=5) mice with treatment of 3% DSS. *P

< 0.05. **P < 0.02. Red asterisk, comparison between the wild-type and Lrrk2−/−BM

recipients; black asterisk, comparison between 1:1 mix and wild-type BM recipients.

Data are representative of two independent experiments (mean and SEM).

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 5

Supplementary Fig. 5. LRRK2 inhibits nuclear translocation of NFAT. Immunoblot

analysis of cytosolic (Cyt) and Nuclear (Nucl) fractions from 293T cells transfected with

NFAT1-GFP (a), HA-NFAT2 (b), HA-NFAT3 (c) and HA-NFAT4 (d) along with vector

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control or Myc-LRRK2 after 1 µM ionomycin (Iono) treatment for 30 min. Hsp90 and

PARP were used as controls for the purity of cytosolic and nuclear fractions,

respectively. Densitometry of each NFAT band is indicated at the bottom of the blots.

The relative intensity of NFAT bands is indicated at the bottom of the blots, presented as

relative to that in cytosolic fraction without Myc-LRRK2 untreated with Iono. Data are

representative of two independent experiments.

Supplementary Figure 6

Supplementary Fig. 6. LRRK2 interacts with multiple components in the NRON

complex. Immunoprecipitation (IP) and immunoblot analysis of 293T cells transfected

with a plasmid expressing myc-LRRK2. Iso, isotype control for immunoprecipitation.

Sizes of proteins are indicated on the left. Data are representative of three independent

experiments.

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 7

Supplementary Fig. 7. LRRK2 inhibition on NFAT1 depends on NRON. (a) Left,

immunoprecipitation (IP) and immunoblotting analysis of 293T cells transfected with

Myc-LRRK2 and NFAT1-GFP with NRON knockdown or nonspecific control (NS), and

right, quantitative real-time PCR analysis (mean ± SEM of triplicates) of NRON,

presented as the percent of that of NS sample. Actin was used for total mRNA

normalization (B) Immunoblot analysis of cytosolic and nuclear fractions of 293T cells

transfected with Myc-LRRK2 and NFAT1-GFP with NRON knockdown or nonspecific

control treated or untreated with 1 µM Iono for 30 min. Data are representative of two

independent experiments.

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 8

Supplementary Fig. 8. Cytokine production by wild-type and Lrrk2−/− macrophages.

ELISA analysis (mean ± SEM of triplicates) of IL-6 (a) and IL-12p40 (b) secretion in

supernatants in BMDMs cultures stimulated for 18 hours with 5 µg/ml flagellin, 10

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µg/ml E. coli single stranded DNA (ssDNA), 2.5 µM oligodeoxynucleotides (ODN)

control, 2.5 µM ODN 1585, 2.5 µM polyinosinic-polycytidylic acid (poly I:C), 10 µg/ml

lipopolysaccharide (LPS) and 10 µM muramyl dipeptide (MDP). (c) Quantitative real-

time PCR analysis (mean ± SEM of triplicates) of IL-6 and IL-12p40 in purified

peritoneal macrophages from WT and Lrrk2−/− 6 hours after intraperitoneally receiving1

mg zymosan (left) or 1 mg Pam3CSK4 (right), presented as relative to actin levels. N.D.

not determined. Data are representative of three independent experiments.

Supplementary Figure 9

Supplementary Fig. 9. LRRK2 deficiency does not affect NF-κB and MAPK activation

by zymosan. Immunoblotting analysis of p-IκBα, p-JNK, p-Erk1/2 and p-p38 in wild-

type and Lrrk2−/− BMDM cells treated with 100 µg/ml zymosan for 0-120 min. Hsp90

was used as a loading control for cell lysates. Data are representative of three

independent experiments.

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 10

Supplementary Fig. 10. CsA treatment suppresses the worsened colitis in Lrrk2−/− mice.

(a) Left, mean body weight as a percent of starting weight and right, mean clinical

scores of wild-type and Lrrk2−/− receiving 3% DSS with or without 1 mg/mouse/day CsA

treatment (n=5 per group). **P < 0.02. P values were calculated between vehicle treated

and CsA treated groups. (b) Left, immunohistochemistry analysis of NFAT1 (brown) in

paraffin-embedded colonic sections from Lrrk2−/− (n=5 per group) mice receiving vehicle

(Mock) or CsA treatment as in (a). Nuclei were counterstained with hematoxylin (blue).

Nature Immunology doi:10.1038/ni.2113

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Scale bar, 25 µm. Right, percent of cells with nuclear NFAT1 among approximately 100

NFAT1-positive cells for each group. Data are representative of two independent

experiments (mean and SEM in a).

Supplementary Figure 11

Supplementary Fig. 11. LRRK2 mRNA levels are not significantly different in

peripheral lymphocytes from 2397M/M and 2397T/T carriers. Quantitative RT-PCR analysis

of LRRK2 and actin mRNA levels in human peripheral B cells, presented as the ratio

between LRRK2 and actin. N.S. not significant. Data are representative of two

independent experiments (mean and SEM).

Nature Immunology doi:10.1038/ni.2113

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Supplementary Figure 12

Supplementary Fig. 12. Cytokine production in primary macrophages from 2397M/M and

2397T/T carriers. Quantitative RT-PCR analysis of IL-6 (a) or IL-12p40 (b) mRNA levels

in primary macrophages stimulated with 50 µg/ml zymosan (Zym) or 5 µg/ml

Pam3CSK4 (Pam) for 2 hours, presented as the ratio of IL-6 or IL-12p40 to actin. Each

symbol represents an individual. N.S. not significant. Data are pooled analysis of one

experiment (mean and SEM).

Nature Immunology doi:10.1038/ni.2113