Supporting InformationMatarese et al. 10.1073/pnas.1210644110SI Materials and MethodsFlow Cytometric Analysis. FITC-conjugated mAbs to CD4, CD11c,Ter, and natural killer cells; PE-conjugated mAbs to CD14, VLA-4 (CD49d), ICAM-1 (CD54), CD25, and CD71; Cy5-conjugatedmAbs to CD3 and Sca-1; and allophycocyanin-conjugated mAbsto CD117 were all purchased from BD Biosciences-Pharmingen,and allophycocyanin-conjugated mAbs to CD19 and CD8 werepurchased from Miltenyi Biotech. Anti-FoxP3 (eBiosciences),PCNA (Chemicon International), and phosphor-S6 (Cell Sig-naling Technology) antibodies were used for intracellular stain-ing, according to manufacturer’s instructions. Incorporation ofbromodeoxyuridine (BrdU) and FACS analysis were performedwith the BrdU Flow Kit (BD-Pharmingen) in accordance withthe manufacturer’s instructions. Isolated thymus, bone marrow,spleen, and lymph node were prepared for flow cytometry byincubating cells with the appropriate Abs or control isotype-matched Abs followed by PBS washes. Flow cytometry experi-ments were performed with a FACSCanto (Becton-Dickinson)and a Dako CyAn (Dako Cytomation) and analyzed by FlowJosoftware (Tree Star Inc.). Immune phenotype of AgRP-Sirt1 KOmice and their littermate controls were performed in basalconditions (naïve mice) and 7 d later antigen immunization withCFA was performed (Difco; BD Diagnostic Systems).

Proliferation Assays. Spleen and lymph node cells were obtainedfrom both strains of mice, dissociated into single-cell suspension,and cultured for proliferation assays in flat-bottomed, 96-wellmicrotiter plates (BDFalcon) at a density of 5× 105 viable cells perwell in a total volume of 200 μL of RPMI-1640 medium (In-vitrogen Corp.) supplemented with 2% FCS (Invitrogen Corp.),2 mM L-glutamine (Invitrogen Corp.), 0.1 mM nonessentialamino acids (Invitrogen Corp.), 1 mM sodium pyruvate (In-vitrogen Corp.), 50 μM 2-mercaptoethanol (Sigma-Aldrich), and100 U/mL penicillin and 100 μg/mL streptomycin (InvitrogenCorp.). Cells were cultured at 37 °C in 100% humidity and 5%CO2 in the presence or absence of either mouse anti-CD3 or anti-CD28 beads (Dynal; Invitrogen) (0.5 bead per cell; 5 × 104 cellsper well) in the case of highly purified T cells or with soluble anti-CD3 mAb stimulation (2C11, 0.5 μg/mL final concentration; BDBiosciences-Pharmingen) in the case of lymph node-derivedlymphocytes, costimulation being provided by the antigen-pre-senting cells (B cells) present in the assay. The Treg:Teff ratio inthe suppression experiments was 1:1. For antigen-specific pro-liferation during EAE, lymph node cells, obtained 20 d afterMOG35–55 immunization from both groups of mice, were culturedin the presence of MOG35–55 peptide at the dose of 50 μg/mL.After 48 h cells were incubated for an additional 16 h with 0.5 μCiper well of [3H]thymidine (Amersham Pharmacia Biotech), har-vested on glass-fiber filters using a Tomtec (Orange) 96-well cellharvester, and counted in a 1205 Betaplate liquid scintillationcounter (Wallac). Results obtained from triplicate cultures areexpressed as mean counts per minute ± SD.

Cytokine Measurement. The mouse Th1/Th2 10 plex Kit (BenderMedSystem) was used according to the manufacturer’s in-structions to measure IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, TNF-α,IFN-γ, and TGF-β production in the cell supernatants by purifiedT-cell subsets (Treg and Teff) or unfractionated lymph node-derived lymphocytes cultured and stimulated as described in theprevious paragraph.

Western Blots and Biochemical Analyses. Total cell lysates wereobtained in 50 mMHepes (pH 7.5), 250 mMNaCl, 1 mM EDTA,1.0% Triton X-100, 10 mM sodium fluoride, 1 mM sodiumorthovanadate, and 2 μg/mL each of aprotinin, leupeptin, andpepstatin. Total proteins (50 μg) from each lysate were subjectedto SDS/PAGE under reducing conditions. After electrophoresis,proteins were transferred onto a nitrocellulose filter membrane(Protan; Schleicher & Schuell BioScience) using a Trans-Blot Cell(Bio-Rad Laboratories) and transfer buffer containing 25 mMTris, 192 mM glycine, and 20% methanol. Membranes wereplaced in 5% nonfat milk in PBS plus 0.5% Tween 20 (PBST) at4 °C for 2 h to block the nonspecific binding sites. Filters wereincubated with specific Abs before being washed three times inPBST and then incubated with a peroxidase conjugated secondaryAb (Amersham Biosciences). After further washing with PBST,peroxidase activity was detected by using the ECL system(Amersham Biosciences). The antibodies used were the fol-lowing: anti–p27Kip-1, anti–phospho-S6, anti-S6, andmouse anti-Sirt1 (all from Cell Signaling Technology) and anti-ERK1/2 andanti–phospho-ERK1/2 (Santa Cruz Biotechnology Inc.). The fil-ters were also probed with a tubulin antibody (Sigma) to normalizefor the amount of loaded protein. All filters were quantified bydensitometric analysis of the bands using the program ScionImage(version 1.63 for Macintosh; Scion Corp. Inc.).

In Vivo Treatment with Propranolol and SR59230A. AgRP-Sirt1 KOand control littermate mice received i.p. injections of propranolol(Sigma; 10 mg·g body weight–1·d–1) and SR59230A (Sigma; 5mg·g body weight–1·d–1) for 3 d. Two hours after the final in-jection, mice were killed and various peripheral tissues wereharvested for analysis.

Real-Time PCR Analysis.Tissue RNA samples were prepared by theTRIzol method (Invitrogen). For real-time PCR analysis, RNAwas reverse-transcribed using the IScript cDNA synthesis kit(BioRad) and used in quantitative PCR reactions containingSYBR-green fluorescent dye (ABI). Relative expression ofmRNAs was determined after normalization withTATA-binding protein (TBP) levels using the ΔΔ Ct method. Quan-titative PCR was performed using the ABI-9300 PCR machine.As a point of reference, the Ct values for both PRDM16 andTBP mRNA expression in BAT were typically 24–26. Student ttest was used for comparisons and to obtain statistics. Primersused for UCP1 mRNA real-time PCR were as follows: Forward5′ ACT GCC ACA CCT CCA GTC ATT; Reverse 5′ CTT TGCCTC ACT CAG GAT TGG.

Matarese et al. www.pnas.org/cgi/content/short/1210644110 1 of 7

Teff

Sirt1

Tubulin

Agrp-Sirt1

KO

WT

Sirt1

/Tub

ulin

WTAgrp-Sirt1 KO

0

0.5

1.0

1.5

Treg

Sirt1

Tubulin

WT Agrp-Sirt1

KO

Sirt1

/Tub

ulin

WTAgrp-Sirt1 KO

0

0.5

1.0

1.5

a

b

Fig. S1. Sirt1 expression in highly purified Teff and Treg cells. (A and B) Immunoblot for Sirt1 and tubulin on purified Teff or Treg cells from WT and AgRP-Sirt1 KO mice, respectively. The graphs show the relative densitometric protein quantification of Sirt1 specifically of the gels shown in WT (black columns) andAgRP- Sirt1 KO (gray columns) mice. One representative out of three independent experiments.

3.5% 4.2%

3.8% 3.5%

SSC

CD3

CD11c

WT Agrp-Sirt1 KO

11.2% 15.3%

Ter

73% 69%

2.1% 2.7%

6.9% 6.5%

SSC

CD34

Sca-1

CD117

WT Agrp-Sirt1 KO

2.8% 3.3%

CD71

26.6% 28.1%

CD19

0

2

4

6

8

n. of

cells

(x10

6 )

WTAgrp-Sirt1 KO

a

b

Fig. S2. Ex vivo bone marrow phenotype of naive AgRP-Sirt1 KO mice and their control littermates. (A) Cell numbers of bone marrow-derived cells in WT(black column) and AgRP-Sirt1 KO (gray column) mice. Data are shown as mean ± SD (n = 5 mice/group). (B) Immune phenotype of WT and AgRP-Sirt1 KO mice.One representative out of three independent experiments.

Matarese et al. www.pnas.org/cgi/content/short/1210644110 2 of 7

42.2% 41.6%

77% 80.1%

23.1% 25.2%SS

C

CD3

CD4

CD8

WT Agrp-Sirt1 KO

0

2

4

6

8

10

n. of

cells

(x10

6 )

WTAgrp-Sirt1 KO

a b

Fig. S3. Ex vivo lymph node phenotype of naive AgRP-Sirt1 KO mice and their control littermates. (A) Cell numbers of lymphocytes in WT (black column) andAgRP-Sirt1 KO (gray column) mice. Data are shown as mean ± SD (n = 5 mice/group). (B) Immune phenotype of WT and AgRP-Sirt1 KO mice. One representativeout of three independent experiments.

SSC

CD25

CD54

WT Agrp-Sirt1 KO

23.1% 19.8%

12.2% 10.9%

23.7% 21.8%

Gated on CD4+

SSC

CD25

CD54

WT Agrp-Sirt1 KO

4.4%

8.8% 9.2%

5.1% 5.9%

Gated on CD8+

6.1%

Ex vivo

CD49d CD49d

Fig. S4. Ex vivo immune phenotype of activation markers from lymph nodes of naive AgRP-Sirt1 KO and WT mice. One representative out of three in-dependent experiments.

Matarese et al. www.pnas.org/cgi/content/short/1210644110 3 of 7

6.1%

5.9%

14%

13%W

TAg

rp-S

irt1 K

O

FoxP

3

CD4 (gated on CD4+)

FoxP

3

CD4 (gated on CD4+)

Ex vivo

Relat

ive ce

ll num

ber

PCNA (gated on CD4+FoxP3+)

PCNA (gated on CD4+FoxP3+)

Relat

ive ce

ll num

ber

Fig. S5. Ex vivo analysis of the percentage of Treg andPCNAexpression in Treg cells from the lymph nodes of naïveAgRP-Sirt1 KOandWTmice. (n= 3mice/group,one representative out of three independent experiments).

Matarese et al. www.pnas.org/cgi/content/short/1210644110 4 of 7

21% 19.6%

34.2% 33.8%

12.7% 11.8%

SSC

CD3

CD4

CD8

WT Agrp-Sirt1 KO

0.7% 0.8%

1.8% 1.9%

SSC

CD14

CD19

CD11c

WT Agrp-Sirt1 KO

45.6% 47.1%

NK

4.0% 4.7%

0

20

40

60

80

n. of

cells

(x10

6 )

WTAgrp-Sirt1 KO

a

b

Fig. S6. Ex vivo spleen phenotype of naive AgRP-Sirt1 KO mice and their control littermates. (A) Cell numbers of splenocytes in WT (black column) and AgRP-Sirt1 KO (gray column) mice. Data are shown as mean ± SD (n = 5 mice/group). (B) Immune phenotype of AgRP-Sirt1 KO and WT mice. One representative outof three independent experiments.

Matarese et al. www.pnas.org/cgi/content/short/1210644110 5 of 7

SSC

CD25

CD54

WT Agrp-Sirt1 KO

CD49d

16.9% 17.4%

23.4% 19.9%

13.7% 14.5%

Gated on CD4+

SSC

CD25

CD54

WT Agrp-Sirt1 KO

CD49d

3.09%

7.2% 6.9%

5.6% 4.8%

Gated on CD8+

3.3%

Ex vivo

Fig. S7. Ex vivo immune phenotype of activation markers from spleens of naive AgRP-Sirt1 KO mice and WT mice. One representative out of three in-dependent experiments.

4.2%

4.0%

11%

12%

WT

Agrp

-Sirt1

KO

FoxP

3

CD4 (gated on CD4+)

FoxP

3

CD4 (gated on CD4+)

Ex vivo

Relat

ive ce

ll num

ber

PCNA (gated on CD4+FoxP3+)

Relat

ive ce

ll num

ber

PCNA (gated on CD4+FoxP3+)

Fig. S8. Ex vivo analysis of the percentage of Treg and PCNA expression in Treg cells from the spleens of naïve AgRP-Sirt1 KO and WT mice (n = 3 mice/group,one representative out of three independent experiments).

Matarese et al. www.pnas.org/cgi/content/short/1210644110 6 of 7

TeffTregTeff/Treg

Agrp-Sirt1 KOWT0

5000

10000

15000

20000

Proli

ferati

on(cp

m)

*** *

Anti-CD3/CD28

IL-2 (

pg/m

l)

Agrp-Sirt1 KOWT

*NS

0

100

200

300

400 * *

0

100

200

300

400

IIL-1

7 (pg

/ml)

*NS

Agrp-Sirt1 KOWT

**

IFN-γ

(pg/m

l)

Agrp-Sirt1 KOWT0

2000

4000

6000

8000 * * *

Fig. S9. Proliferation and IL-2, IL-17, and IFN-γ production of purified Teff, Treg cells, and of both cell types in coculture from WT and AgRP-Sirt1 KO mice,upon anti-CD3/CD28 stimulation. The data are shown as mean ± SD (n = 5 mice/group; *P < 0.0001, **P < 0.05).

Fig. S10. UCP1 mRNA expression in brown adipose tissue of WT and AgRP-Sirt1 KO mice with and without treatment with propanolol and SR59230A. UCP1levels in mutant mice were inhibited by sympathetic blockade. Data are shown as mean ± SD (n = 5 mice/group; *P < 0.05).

Matarese et al. www.pnas.org/cgi/content/short/1210644110 7 of 7