Supplementary Figure 1 - Nature

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5. Gating strategies a. Sequential gating strategy for sorting SP, DP and DN .... Brefeldin A, 2 μM Monensin and 2.5 μg/ml FITC-labeled anti mouse CD107a. ..... 1606 RNA polymerase II, heptapeptide repeat, - PF05001. 1610 .... GI:339895989.
Supplementary Figure 1 b 0.015 NS

0.010

0.005

0.000 Thymic stromal cells (n=3)

0.015

Relative expression (Ratio of ARMC5/ -actin)

Rel at i ve expression (Ratio of ARMC5/ -actin)

a

0.010

0.005

0.000

DN1 (n=3)

Thymocytes (n=3)

DN2 (n=3)

DN3 (n=3)

DN4 (n=3)

NS Relative expression

0.010 0.008 0.006 0.004

0.002 0.000 DP

DN

CD4+

CD8+

(n=3)

(n=3)

(n=3)

(n=3)

(Ratio of ARMC5/ -actin)

d Relative expression (Rat io of ARMC5/ -actin)

c

NS

0.025

NS

0.020 0.015

0.010 0.005 0.000 Naive (N=3)

Memory (N=3)

Armc5 mRNA expression in different thymocyte and T-cell subpopulations RNA was extracted from different subpopulations of thymocytes and T cells, or from T cells cultured under different conditions. Armc5 mRNA expression levels in these cells were measured by RT-qPCR, with -actin mRNA levels as internal controls. The numbers (n) of experiments performed are indicated. Pooled results of multiple experiments are expressed as means ± SEM of ratios of Armc5 versus -actin signals, unless specified otherwise. Two-tailed Student’s t-test was used in data between two groups. One way ANOVA followed with Bonferroni’s multiple comparisons test was used in data among four groups. NS, no significance.

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a. Armc5 mRNA expression in thymocytes and thymic stroma cells Thymocytes were flushed out from the thymus of WT mice, and the remainder was considered to be thymic stroma cells. b-c. Armc5 expression in thymocyte subpopulations Thymocyte subpopulations (B: DN1-4; C: CD4 SP, CD8 SP; CD4CD8 DP, and DN) were sorted by flow cytometry. d. Armc5 mRNA expression in naïve versus memory T cells CD62L+CD44lo naïve T cells and CD62L+CD44int-hi memory T cells were sorted by flow cytometry from WT spleen cells.

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Supplementary Figure 2

3

Supplementary Figure 2 continued

4

Gating strategies a. Sequential gating strategy for sorting SP, DP and DN (DN1-4) populations in thymocytes. b. Sequential gating strategy for sorting memory cells (CD62L+CD44int-hi) and naïve cells (CD62L+CD44lo) in Thy1.2+ lymph nodes cells. c. Sequential gating strategy for CD4+ or CD8+ cells in spleen cells. d. Sequential gating strategy for B220+ cells in spleen cells. e. Sequential gating strategy for the cell cycle analysis of T cells. f. Sequential gating strategy for apoptosis analysis of CD4+ and CD8+ cells. g. Sequential gating strategy for CD4+/IL17+ or CD4+/IFN-+ cells. h. Sequential gating strategy for CD4+ cells derived from WT and KO donor cells in chimeric mice. i. Sequential gating strategy for CD4+ cells. j. Sequential gating strategy for CD4+ or CD8+ cells in LCMV infection mice model. k. Sequential gating strategy for CD8+ and tetramers+ cells in LCMV infection mice model.

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Supplementary Figure 3

Mouse number

1 2 3 4 5 6 7 +/- +/- +/+ +/- +/- +/- +/+

9.3 kb 6.6 kb

EcoRV-5’ probe +/- +/- +/+ +/- +/- +/- +/+

12 .5 kb 8.7 kb

Hind III-5’ probe

Genotyping of Armc5 mutant mice Tail DNA was digested with EcoRV and analyzed by Southern blotting (top panel) with the 5’ probe whose location is indicated (Fig. 2a). A 9.3-kb band representing the WT allele and a 6.6-kb band representing the recombinant allele are shown. Similarly, tail DNA was digested with HindIII and analyzed with the 3’ probe (bottom panel). A 12.5kb band representing the WT allele and an 8.7-kb band representing the recombinant allele are indicated.

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Supplementary Figure 4 Females NS

160

Growth hormone (ng/ml)

Growth hormone (ng/ml)

Males

120 80 40

0

K O (n= 6)

WT (n= 6)

200

NS

150 100 50 0 K O (n= 5)

WT (n= 5)

Serum growth hormone levels in KO mice Serum growth hormone levels in 8-12-week-old KO and WT mice were measured by ELISA. The results are reported as scatter plots, with each symbol representing actual values. Mouse numbers (n) in each group are indicated. p>0.05 (two-tailed Student’s t test). NS: not significant.

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Supplementary Figure 5

KO

WT

Adrenal gland histology of young KO mice Adrenal glands from WT and KO mice (8-12 weeks old) were sectioned and stained with H/E. Representative micrographs from a KO (12-week-old male) mouse and its WT male littermate. No histological abnormalities were found in the KO adrenal gland.

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Supplementary Figure 6

NS

Glucocorticoids (ng/ml)

10

8 6

4 2 0 W T (n =1 0)

K O (n =6)

Serum glucocorticoid levels in young WT and KO mice The mice were bled between 12:30-1:30 pm. Serum levels (means ± SEM) of glucocorticoids old KO and WT mice are shown. Two-tailed Student’s t test was used for statistical analysis. NS: not significant.

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Supplementary Figure 7 NS

0.06 0.04

0.02 0.00

W T (n =5)

b

NS

0.10 0.08 0.06 0.04 0.02 0.00

W T (n =5 )

c

WT

NS

80 60 40

20 0

K O (n =5 )

KO Sp leen weight (g )

WT

Thymus cellularity per lobe (x106)

KO

K O (n =5 )

W T (n = 4)

S pleen cellularity (x106)

WT

Th ym u s weight (g )

a

K O (n =4)

NS

200 150 100

50 0

W T (n =4)

K O (n = 4)

KO 74.5

3.0

78.0

4.6

16.3

4.6

14.4

CD8

4.6

CD4

d

WT

KO

WT

KO

44.3

43.8

33.9

CD8

B220

37.4

42.6

56.5

38.5

Thy1.2

59.5

CD4

Thymus and spleen weight, cellularity and cell subpopulations in KO mice a. thymus size, weight, and cellularity in WT and KO mice Left panel: Representative photo of the KO and WT thymus from 8-week-old littermates. Right panels: thymus weight and cellularity of KO and WT from 8-12-week-old male littermates. Mouse numbers (n) in each group (n) are indicated. p>0.05 (2-tailed Student’s t test). NS: not significant. 10

b. spleen size, weight, and cellularity in WT and KO mice Left panel: Representative photo of KO and WT spleen from 8-week-old littermates. Right panels: weight and cellularity of KO and WT spleen from 8-12-week-old male littermates. Mouse numbers (n) in each group (n) are indicated.

p>0.05 (2-tailed

Student’s t test). c. T-cell subpopulations in KO thymus in WT and KO mice Thymocytes from adult KO and WT mice (8-12 weeks old) were analyzed by flow cytometry for percentages of CD4+, CD8+, and CD4+CD8+ subpopulations. Experiments were conducted more than 3 times. Representative dot plots are reported. d. Cell subpopulations in WT and KO Spleen Spleen cells from adult KO and WT mice (8-12 weeks old) were analyzed by flow cytometry for percentages of Thy1.2+ T cells versus B220+ B cells (left panel), and CD4+ versus CD8+ cells (right panel). Experiments were conducted more than 3 times, and representative dot plots are shown.

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Supplementary Figure 8

CD25 and CD69 expression in CD4+ and CD8+ cells after anti-CD3 stimulation Spleen cells were stimulated with anti-CD3mAbg/ml for 16 hours. The cells were gated on CD4-positive and CD8-positive. Experiments were conducted independently 3 times. Representative dot plots are shown.

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Serum total IgG (μ g/mL)

Supplementary Figure 9 400 NS 300 200

100 0

WT (n=7)

KO (n=7)

Serum IgG levels in WT and KO mice Total IgG levels in WT and KO mouse sera were measured by ELISA, and means ± SEM are presented. Mouse numbers (n) in each group are indicated. No statistically significant difference between WT and KO IgG levels was observed (2-tailed Student’s t test). NS: not significant.

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Supplementary Figure 10 White blood cells WT KO

a

13.5%

82.8%

WT

11.8%

KO

68.3% 30.9%

CD45.2

CD45.1

CD45.1 Thy1.2 + T cells WT KO 30.2%

63.8%

WT 35.3%

68.0%

CD45.2

69.0%

CD8+ T cells

d

KO

31.4%

64.9%

33.9%

CD45.2

c

63.6% 35.0%

CD45.2

82.5%

CD4+ T cells

b

CD45.1

CD45.1

Implantation of donor cells in blood and spleen of chimeric mice Eight weeks after KO and WT fetal liver transplantation, peripheral blood cells from recipient mice were examined by flow cytometry. Percentages of donor-derived (CD45.2 single-positive cells) versus recipient-derived (CD45.1/CD45.2 double-positive cells; panel a), total T cells (Thy1.2+ cells; panel c), CD4 cells (panel b) and CD8 cells (panel d) in the spleen were measured by flow cytometry. Experiments were conducted more than 4 times, and representative dot plots are shown.

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Supplementary Figure 11

Multiple parameters in spleen T cells on day 8 post-LCMV infection WT and KO spleen T cells were analyzed for different parameters by flow cytometry on day 8 post-LCMV infection. Representative flow cytometric dot plots are shown (pooled results of the experiments are summarized in bar graphs and presented in Figure 7 in the text proper).

a - b. Activation of LCMV-specific spleen CD8 T cells 15

On the left panel, gp33-41, np396-405 and gp276-286 tetramer-positive CD8 cells in the KO and WT mouse spleens were assessed for activation markers (CD62LloCD44hi), and the percentages of this effector memory cell subpopulation are indicated. On the right panel, the dot plot shows the percentages of this population gated on total CD8 cells. c - d. Expression of IFN- and/or TNF- in gp33-41-stimulated CD8 and gp61-80-stimulated CD4 cells Dot plots show intracellular IFN- and TNF-expression in WT and KO CD8 (B) and CD4 cells (C) stimulated by gp34-41 (for CD8 cells) and gp61-80 (for CD4 cells) (both at 5 M), respectively, for 5 h, in the presence of 50 U/ml IL-2, 5 g/ml Brefeldin A and 2 M Monensin. e. gp33-41-specific CD107a+GranB+ CD8 T cells on day 8 post-LCMV infection Spleen cells from KO and WT mice on day 8 post-LCMV infection were stimulated ex vivo with gp33-41 peptide (5 M) for 5 h in the presence of 50 U/ml IL-2, 5 g/ml Brefeldin A, 2 M Monensin and 2.5 g/ml FITC-labeled anti mouse CD107a. Percentages of CD107a+ and GranB+ cells among CD8 cells were quantified by flow cytometry.

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Supplementary Figure 12 Dom Sight : HTH_RP1_hgx4009v1 vs. Hum an Thym ocyt es (CD4+ , CD8+ ) RP1 (19 Jun 2015) (Bait p lasm id (s): h g x 400 9 v 1 _p B2 9 ) B a it f r a g m e n t SID f r a g m e n t Pf a m o r SM A RT d o m a in Tr a n sm e m b r a n e d o m a in ( TMHM M ) Coi le d -c oi l d o m a in ( N c oi ls) Sig n a l p e p t id e ( Sig n a lPHM M )

SID : Se le c t e d In t e r a c t io n D o m a in It is t h e a m in o a c id se q u e n c e sh a r e d b y a ll p r e y f r a g m e n t s m a t c h in g t h e sa m e r e f e r e n c e p r o t e in . SID s h a v e b e e n f o u n d in n u m e r o u s c a se s t o c o r r e sp o n d t o a n id e n t if ie d st r u c t u r a l o r f u n c t io n a l d o m a in .

Legend

1 ARMC5 Hom o sap ien s GID: 1 5 7 4 2 6 8 5 5 Gen e ID: 7 9 7 9 8

B

D

D

C

A

935

30 1 1

935 35 22

SID Sig n al p ep t id e d om ain 141 183 184 226

Arm ad ill o - SM0 0 1 8 5 225 Ar m ad ill o - SM0 0 1 8 5 267 Ar m ad ill o - SM0 0 1 8 5 358 403

Arm ad ill o - SM0 0 1 8 5

1 ARMC5 Hom o sap ien s GID: 1 5 7 4 2 6 8 5 5 Gen e ID: 7 9 7 9 8

935

30 1 1

935 35 22

SID Sig n al p ep t id e d om ain 141 183 184 226

Arm ad ill o - SM0 0 1 8 5 225 Ar m ad ill o - SM0 0 1 8 5 267 Ar m ad ill o - SM0 0 1 8 5 358 403

1 C1 0 orf4 6 Hom o sap ien s GID: 1 0 9 4 5 2 5 9 6 Gen e ID: 1 4 3 3 8 4

hgx4009v1

Arm ad ill o - SM0 0 1 8 5

369

28

270 258

137

SID Cu ll in , N-t erm in al - PF0 0 8 8 8

1

454

CDCA7 L v ar 1 Hom o sap ien s GID: 2 2 6 3 7 1 6 7 1 Gen e ID: 5 5 5 3 6

125

454 443

344

SID Z in c-fin g er d om ain of m on oam in e-ox id ase - PF1 0 4 9 7

1 CUL3 Hom o sap ien s GID: 3 8 0 7 1 4 6 6 1 Gen e ID: 8 4 5 2

hgx4009v1

768

19 34

376

SID

413

563

666 Cu ll in , N-t er m in al - PF0 0 8 8 8 Cu ll in h om ol og y - SM0 0 1 8 2 695 761 Cu ll in p r ot ein , n ed d y lat ion d om ain - PF1 0 5 5 7 695 762 Cu ll in p r ot ein , n ed d y lat ion d om ain - SM0 0 8 8 4

1 DAPK1 Hom o sap ien s GID: 8 9 3 6 3 0 4 7 Gen e ID: 1 6 1 2

1430 1072

13 13

275 275

1237

SID

Pr ot ein k in ase, cat aly t ic d om ain - PF0 0 0 6 9 Serin e/t h reon in e- / d u al sp ecificit y p r o - SM0 0 2 2 0 378 407 An k y rin rep eat - SM0 0 2 4 8 379 410 An k y rin rep eat - PF0 0 0 2 3 411 440 An k y r in rep eat - SM0 0 2 4 8 414 464 An k y r in rep eat s (m an y cop ies) - PF1 3 6 3 7 444 473 An k y r in r ep eat - SM0 0 2 4 8 477 506 An k y r in r ep eat - SM0 0 2 4 8 479 508 An k y r in rep eat - PF0 0 0 2 3 510 539 An k y r in rep eat - SM0 0 2 4 8 511 542 An k y r in rep eat - PF0 0 0 2 3 543 572 An k y r in rep eat - SM0 0 2 4 8 543 574 An k y r in r ep eat - PF0 0 0 2 3 576 607 An k y rin rep eat - PF0 0 0 2 3 576 605 An k y r in r ep eat - SM0 0 2 4 8 609 640 An k y r in rep eat - PF0 0 0 2 3 609 638 An k y rin rep eat - SM0 0 2 4 8 642 671 An k y rin rep eat - SM0 0 2 4 8 1299 1312

1

D

E2 F2 Hom o sap ien s GID: 4 7 5 8 2 2 6 Gen e ID: 1 8 7 0

D

FAM6 5 B Hom o sap ien s GID: 1 6 4 4 1 4 4 2 1 Gen e ID: 9 7 5 0

D

D

D

1396 1393

Deat h d om ain - SM0 0 0 0 5 Deat h d om ain - PF0 0 5 3 1

437

16 129

271 SID Tr an scr ip t ion fact or E2 F/d im er isat ion p a - PF0 2 3 1 9

194

1

1068 456 89

109

764

SID

Coi led d om ain 773

793

Coi led d om ain

1

1250

FLJ2 0 1 0 5 Hom o sap ien s GID: 5 8 3 3 1 2 6 7 Gen e ID: 5 4 8 2 1

2

1

103

H4 FJ Hom o sap ien s GID: 2 1 2 6 4 6 0 0 Gen e ID: 8 3 6 7

22 16 28

103 90 94

370 53

73

SID

Coi led d om ain

92 99

289

Helicase, su p erfam ily 1 /2 , ATP-b in d in g d - SM0 0 4 8 7 416 SNF2 -r elat ed - PF0 0 1 7 6 490 574 Helicase, C-t erm in al - SM0 0 4 9 0 495 574 Helicase, C-t erm in al - PF0 0 2 7 1

SID Hist on e H4 - SM0 0 4 1 7 Hist on e cor e - PF0 0 1 2 5

1

1268

HDLBP Hom o sap ien s GID: 4 8 8 5 4 0 9 Gen e ID: 3 0 6 9

1071 274

294

490

510

217 210 221 225

SID

Coi led d om ain

776

149 153

1268

Coi led d om ain

803 849

Coi led d om ain 873 Coi led d om ain 1024

1044 Coi led d om ain K Hom ol og y d om ain - SM0 0 3 2 2 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 289 K Hom ol og y d om ain - SM0 0 3 2 2 284 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 362 K Hom ol og y d om ain - SM0 0 3 2 2 355 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 363 429 K Hom ol og y d om ain - SM0 0 3 2 2 368 424 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 434 502 K Hom ol og y d om ain - SM0 0 3 2 2 439 496 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 506 575 K Hom ol og y d om ain - SM0 0 3 2 2 512 568 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 580 648 K Hom ol og y d om ain - SM0 0 3 2 2 584 643 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 652 721 K Hom ol og y d om ain - SM0 0 3 2 2 656 716 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 726 795 K Hom ol og y d om ain - SM0 0 3 2 2 731 789 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 799 868 K Hom ol og y d om ain - SM0 0 3 2 2 804 863 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 872 972 K Hom ol og y d om ain - SM0 0 3 2 2 879 966 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 973 1039 K Hom ol og y d om ain - SM0 0 3 2 2 974 1033 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 1051 1122 K Hom ol og y d om ain - SM0 0 3 2 2 1055 1117 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3 1126 1195 K Hom ol og y d om ain - SM0 0 3 2 2 1132 1190 K Hom ol og y d om ain , t y p e 1 - PF0 0 0 1 3

294 298

D

1

1499

HUWE1 Hom o sap ien s GID: 6 1 6 7 6 1 8 8 Gen e ID: 1 0 0 7 5

1402 90

370 429

1423

815

E3 u b iq u it in lig ase, d om ain of u n k n ow n f - PF0 6 0 2 5 1317 1318

1352 1354

Ub iq u it in -associat ed /t r an slat ion elon g at - PF0 0 6 2 7 Ub iq u it in -associat ed /t r an slat ion elon g at - SM0 0 1 6 5

1500

2999 1951

2177

SID 2425

2445

Coi led d om ain 2681

1616

1678

2703

Coi led d om ain

WWE d om ain - PF0 2 8 2 5 2968

3000 3029 3000

Coi led d om ain 3110 Coi led d om ain Dom ain of u n k n ow n fu n ct ion DUF4 4 1 4 - PF1 4 3 7 7

3079

4374 4373

4069

1 IMMT Hom o sap ien s GID: 1 5 4 3 5 4 9 6 3 Gen e ID: 1 0 9 8 9

758 672

1

16

Sig n al p ep t id e d om ain 207

227 281

Coi led d om ain 315 Coi led d om ain 398 425

Coi led d om ain 510 530

758

746

Mit och on d r ial in n er m em b ran e p r ot ein Mit - PF0 9 7 3 1

1 KDM2 A Hom o sap ien s GID: 1 6 3 0 6 5 7 9 Gen e ID: 2 2 9 9 2

1162 148 199

316 299

461 Jm jC d om ain - SM0 0 5 5 8 Jm jC d om ain - PF0 2 3 7 3

622

565

609 619

D

SID

Coi led d om ain

44

E

SID

Z in c fin g er, CXXC-t y p e - PF0 2 0 0 8 676 Z in c fin g er , PHD-t y p e - SM0 0 2 4 9 893 896

933 934

F-b ox d om ain , cy clin -lik e - SM0 0 2 5 6 F-b ox d om ain , cy clin -lik e - PF0 0 6 4 6

1 KIF1 1 Hom o sap ien s GID: 1 9 7 3 0 4 7 9 7 Gen e ID: 3 8 3 2

1056 368

402 412 447

618 Coi led d om ain 439 Coi led d om ain 474 Coi led d om ain 485 505 Coi led d om ain

937

805 16 24

2999

4374 3049 3090

4036

D

Coi led d om ain

E3 u b iq u it in lig ase, d om ain of u n k n ow n f - PF0 6 0 1 2

367 359

825

SID

Coi led d om ain

Kin esin , m ot or d om ain - SM0 0 1 2 9 Kin esin , m ot or d om ain - PF0 0 2 2 5 916

17

1053

Kin esin -associat ed m icr ot u b u le-b in d in g d - PF1 3 9 3 1

HECT - SM0 0 1 1 9 HECT - PF0 0 6 3 2

Dom ain of u n k n ow n fu n ct ion DUF4 4 1 4 - PF1 4 3 7 7

Supplementary Figure 12 continued Dom Sight : HTH_RP1_hgx4009v1 vs. Hum an Thym ocyt es (CD4+ , CD8+ ) RP1 (19 Jun 2015) (Ba it p la sm id (s): h g x 400 9 v 1 _p B2 9 ) Bait fragment SID fragment Pfam or SMART domain Transmembrane domain (TMHMM) Coiled-coil domain (Ncoils) Signal peptide (SignalPHMM)

SID: Selected Interaction Domain It is the amino acid sequence shared by all prey fragments matching the same reference protein. SIDs have been found in numerous cases to correspond to an identified structural or functional domain. Legend

D

B

D

1 M TF2 Ho m o sa p ie n s GID : 2 5 6 4 1 9 0 1 1 Ge n e ID : 2 2 8 2 3

491

30 2 2

429 Z in c f in g e r , PHD -t y p e - SM 0 0 2 4 9 Z in c f in g e r , PHD -f in g e r - PF0 0 6 2 8 151 Z in c f in g e r , PHD -t y p e - SM 0 0 2 4 9 442

53 53 101

491 SID Coi le d d o m a in

490

Po ly co m b -lik e M TF2 f a ct o r 2 , C-t e r m in a l - PF1 4 0 6 1

1

772

NFE2 L1 Ho m o sa p ie n s GID : 1 8 9 1 8 1 6 7 0 Ge n e ID : 4 7 7 9

185

341

1 PCBP1 Ho m o sa p ie n s GID : 2 2 2 3 5 2 1 5 0 Ge n e ID : 5 0 9 3

SID 672 652 656

713 716 714

Coi le d d o m a in Ba sic-le u cin e zip p e r d o m a in - SM 0 0 3 3 8 Ba sic-le u cin e zip p e r d o m a in - PF0 0 1 7 0

356 60

12 15

80 75 96 99

B

449

283 SID K Ho m ol o g y d o m a in - SM0 0 3 2 2 K Ho m ol o g y d o m a in , t y p e 1 - PF0 0 0 1 3 167 K Ho m ol o g y d o m a in - SM 0 0 3 2 2 162 K Ho m ol o g y d o m a in , t y p e 1 - PF0 0 0 1 3 278 348 K Ho m ol o g y d o m a in - SM 0 0 3 2 2 282 343 K Ho m ol o g y d o m a in , t y p e 1 - PF0 0 0 1 3

1

1499

POLR2 A Ho m o sa p ie n s GID : 3 0 6 4 8 2 6 5 4 Ge n e ID : 5 4 3 0

14 29 699

719

949

969

354

246 356

1970

631 88

7

29

196 Tr a n sm e m b r a n e d o m a in

SID 539

561 573 605

1 3

D

B

E

22

22 33

STK2 4 Ho m o sa p ie n s GID : 1 1 0 3 4 9 7 3 8 Ge n e ID : 8 4 2 8

4

Tr a n sm e m b r a n e d o m a in 595 Tr a n sm e m b r a n e d o m a in 622 Tr a n sm e m b r a n e d o m a in

Sig n a l p e p t id e d o m a in

628

1 SERTAD 3 Ho m o sa p ie n s GID : 1 5 7 1 8 6 8 4 Ge n e ID : 2 9 9 4 6

Coi le d d o m a in

15 80 SID 15 94 16 06 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 16 10 16 22 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 16 16 16 29 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 16 23 16 36 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 16 30 16 43 RNA p oly m e r ase II, h e p t ap e p t id e r e p e a t , - PF0 5 0 0 1 16 44 16 57 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 16 58 16 71 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 16 72 16 85 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 16 86 16 99 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 00 17 13 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 14 17 27 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 28 17 41 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 42 17 55 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 57 17 69 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 70 17 83 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 84 17 97 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 17 98 18 11 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 26 18 39 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 41 18 53 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 47 18 60 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 55 18 67 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 61 18 74 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 68 18 81 RNA p oly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 75 18 88 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 82 18 95 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 89 19 02 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 18 96 19 09 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 19 04 19 16 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e at , - PF0 5 0 0 1 19 10 19 23 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 19 24 19 36 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 19 31 19 47 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1 19 41 19 54 RNA p oly m e r ase II, h e p t ap e p t id e r e p e a t , - PF0 5 0 0 1 19 48 19 60 RNA p o ly m e r a se II, h e p t a p e p t id e r e p e a t , - PF0 5 0 0 1

1 RPN2 Ho m o sa p ie n s GID : 2 0 9 4 1 3 7 3 6 Ge n e ID : 6 1 8 5

12 82

RNA p o ly m e r a se Rp b 1 , d o m a in 1 - PF0 4 9 9 7 549 RNA p oly m e r ase , N-t e r m in al - SM0 0 6 6 3 521 RNA p o ly m e r a se , a lp h a su b u n it - PF0 0 6 2 3 525 691 RNA p o ly m e r a se Rp b 1 , d o m a in 3 - PF0 4 9 8 3 718 823 RNA p o ly m e r a se Rp b 1 , d o m a in 4 - PF0 5 0 0 0 830 14 24 RNA p o ly m e r a se Rp b 1 , d o m a in 5 - PF0 4 9 9 8 896 10 79 RNA p o ly m e r a se Rp b 1 , d o m a in 6 - PF0 4 9 9 2 11 64 12 99 RNA p o ly m e r a se Rp b 1 , d o m a in 7 - PF0 4 9 9 0

1500 15 00

D

Rib op h o r in II - PF0 5 8 1 7

196 196 SERTA - PF0 6 0 3 1

70

SID

1

431 50

77

140 SID Coi le d d om ain

24 24

274 274

Pr o t e in k in a se , ca t a ly t ic d o m a in - PF0 0 0 6 9 Se r in e /t h r e o n in e - / d u a l sp e cif icit y p r o - SM 0 0 2 2 0

1

1499

TAF1 Ho m o sa p ie n s GID : 2 0 3 5 7 5 8 5 Ge n e ID : 6 8 7 2

10 76

11 44 10 96 11 40

13 13 Coi le d d o m a in 11 60 Coi le d d o m a in 12 34 12 65

SID

Coi le d d o m a in 14 95

26

86

1500

10 48

Tr a n scr ip t io n in it ia t io n f a ct o r TFIID su - PF1 2 1 5 7 13 99 14 11

1893

15 00 15 22 Coi le d d o m a in 1 5 0105 0 7 Br o m o d o m a in - SM 0 0 2 9 7 15 21 16 30 Br o m o d o m a in - SM 0 0 2 9 7 15 34 16 16 Br o m o d o m a in - PF0 0 4 3 9

1

682

TCF1 2 Ho m o sa p ie n s GID : 4 6 3 7 0 0 7 8 Ge n e ID : 6 9 3 8

277

490

SID 578 583

631 636

1

E

TOX4 Ho m o sa p ie n s GID : 9 9 0 7 7 1 1 6 Ge n e ID : 9 8 7 8

B

TT F1 Ho m o sa p ie n s GID : 3 2 8 9 2 7 0 0 3 Ge n e ID : 7 2 7 0

My c-t y p e , b a sic h e lix -lo o p -h e lix (b HLH) - PF0 0 0 1 0 My c-t y p e , b a sic h e lix -lo o p -h e lix (b HLH) - SM 0 0 3 5 3

621 157

442 273 222 223

293 292 291

SID

Coi le d d o m a in Hig h m o b il it y g r o u p (HM G) b o x d o m a in - SM 0 0 3 9 8 Hig h m o b il it y g r o u p (HM G) b o x d o m a in - PF0 0 5 0 5

1

905 430

640

616 621 665

E

SID 663 SANT/M y b d o m ain - SM0 0 7 1 7 677 My b -lik e D NA-b in d in g d o m a in - PF1 3 9 2 1 747 SANT/M y b d o m a in - SM0 0 7 1 7

1 Z BTB3 8 Ho m o sa p ie n s GID : 1 4 8 2 7 6 9 8 9 Ge n e ID : 2 5 3 4 6 1

23 33

14 99

Coi le d d o m a in

TAFII-2 3 0 TBP-b in d in g - PF0 9 2 4 7 586

D

SID

Coi le d d o m a in 12 62

16

14 99

Coi le d d o m a in

1195 127 131

366 BTB/POZ - PF0 0 6 5 1 BTB/POZ -lik e - SM 0 0 2 2 5 342 357 371

622

SID

364 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 382 Z in c-f in g e r d ou b le d om ain - PF1 3 4 6 5 398 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 460 482 Z in c f in g e r , C2 H2 - PF0 0 0 9 6 460 482 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 488 510 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 504 525 Z in c-f in g e r d o u b le d o m a in - PF1 3 4 6 5 516 539 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 10 10 10 32 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 10 25 10 48 Z in c-f in g e r d o u b le d o m a in - PF1 3 4 6 5 10 38 10 60 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 10 52 10 76 Z in c-f in g e r d o u b le d o m a in - PF1 3 4 6 5 10 66 10 88 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 10 81 11 04 Z in c-f in g e r d o u b le d o m a in - PF1 3 4 6 5 10 94 11 16 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 11 25 11 47 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5

18

14 99 14 90

Br o m o d o m a in - SM 0 0 2 9 7 Br o m o d o m a in - PF0 0 4 3 9

Supplementary Figure 12 continued

Dom Sight : HTH_RP1_hgx4009v1 vs. Hum an Thym ocyt es (CD4+ , CD8+ ) RP1 (19 Jun 2015) (Ba it p la sm id (s): h g x 400 9 v 1 _p B2 9 ) Bait fragment SID fragment Pfam or SMART domain Transmembrane domain (TMHMM) Coiled-coil domain (Ncoils) Signal peptide (SignalPHMM)

SID: Selected Interaction Domain It is the amino acid sequence shared by all prey fragments matching the same reference protein. SIDs have been found in numerous cases to correspond to an identified structural or functional domain. Legend

D

1 Z BTB4 0 Ho m o sa p ie n s GID : 1 3 9 3 9 4 5 7 8 Ge n e ID : 9 9 2 3

1239 709

18 24

114 117

885

736

756 766

1

D

Ge n Ma t ch Ho m o sa p ie n s GI:2 8 2 2 1 3 7

1

D

Ge n Ma t ch Ho m o sa p ie n s GI:2 5 6 4 7 5 0

1

D

Ge n Ma t ch Ho m o sa p ie n s GI:3 3 9 8 9 5 9 8 9

1

D

D

D

D

D

235

Ge n Ma t ch Ho m o sap ie n s GI:1 3 3 5 7 3 6 9

116

SID

254

1

254

1 Ge n Ma t ch Ho m o sa p ie n s GI:2 0 3 3 0 7 3 5

137

SID

1

1

D

Ge n Ma t ch Ho m o sa p ie n s GI:1 5 2 1 7 3 9 3

1

D

Ge n Ma t ch Ho m o sa p ie n s GI:1 5 1 4 5 5 8 6

1

SID

137

1

1

SID

116

1

Ge n Ma t ch Ho m o sa p ie n s GI:1 4 9 7 1 1 9 4

D

SID

235

1

D

D

213

1

Ge n Ma t ch Ho m o sa p ie n s GI:2 3 3 0 7 9 3 3

D

SID

213

1 Ge n Ma t ch Ho m o sa p ie n s GI:2 3 9 7 4 0 4 1 7

233

1

194

1

SID

138 138

SID

1

243 214

1

SID

276 276

1

132

SID

331

1

331

1 Ge n Ma t ch Ho m o sa p ie n s GI:1 3 4 4 3 2 7 7

1

99

SID

212

1

212

1 Ge n Ma t ch Ho m o sa p ie n s GI:7 1 6 1 1 8 7

1

185

1

82

SID

232

1

217

1

D

Ge n Ma t ch Ho m o sa p ie n s GI:5 0 4 2 3 8 4

D

Ge n Ma t ch Ho m o sap ie n s GI:1 8 8 5 4 9 5 2

1

D

Ge n Ma t ch Ho m o sa p ie n s GI:1 6 1 6 2 1 4 1 3

1 41

SID

115

1

115

1

SID

225 225

1

SID

293 293 63 70

1

SID

82

1 Ge n Ma t ch Ho m o sa p ie n s GI:1 5 8 8 5 3 0 4 5

SID

185

1 Ge n Ma t ch Ho m o sa p ie n s GI:3 0 7 1 3 3 6 9 6

SID

99

1 Ge n Ma t ch Ho m o sa p ie n s GI:1 3 3 9 2 3 3 6 6

SID

132

1 Ge n Ma t ch Ho m o sap ie n s GI:1 7 3 8 9 9 0 4

SID

162 106

1

D

D

399

1

Ge n Ma t ch Ho m o sa p ie n s GI:7 8 9 9 1 2 9 1 3

D

SID

399

1

D

D

319

1

Ge n Ma t ch Ho m o sa p ie n s GI:1 8 3 0 8 3 4 5

Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 789 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 807 830 Z in c fin g e r , C2 H2 -lik e - SM0 0 3 5 5 809 830 C2 H2 -t y p e zin c f in g e r - PF1 3 8 9 4 836 858 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 851 874 Z in c-f in g e r d o u b le d o m a in - PF1 3 4 6 5 864 887 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 878 903 Z in c-f in g e r d o u b le d o m a in - PF1 3 4 6 5 893 915 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 908 931 Z in c-f in g e r d o u b le d om ain - PF1 3 4 6 5 921 944 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 950 973 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 978 10 00 Z in c fin g e r , C2 H2 - PF0 0 0 9 6 978 10 00 Z in c fin g e r , C2 H2 -lik e - SM0 0 3 5 5 10 06 10 28 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 10 21 10 56 Z in c-f in g e r d o u b le d o m a in - PF1 3 4 6 5 10 46 10 69 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 10 75 10 98 Z in c f in g e r , C2 H2 -lik e - SM0 0 3 5 5 11 04 11 27 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5 11 35 11 58 C2 H2 -t y p e zin c f in g e r - PF1 3 8 9 4 11 35 11 58 Z in c f in g e r , C2 H2 -lik e - SM 0 0 3 5 5

319

1

Ge n Ma t ch Ho m o sa p ie n s GI:1 1 4 1 4 4 8 7

SID

BTB/POZ - PF0 0 6 5 1 BTB/POZ -lik e - SM0 0 2 2 5

41

SID

Tr a n sm e m b r a n e d o m a in 89 Tr a n sm e m b r a n e d o m a in Sig n a l p e p t id e d o m a in

Binding regions between ARMC5 and its associating molecules The cDNA coding sequences representing the protein binding regions between ARMC5 and its preys are illustrated.

19

Supplementary Figure 13 b

a

Normalized relative expression (Ratio of ARMC5/ -actin)

Normalized relative expression (Ratio of ARMC5/ -actin)

Th 1 condition 1.1 1.0 0.9 0.8 0.7 0.6

0.5 0

1

2

3

Days

Th17 condition 1.1 1.0 0.9 0.8 0.7 0

1

2

3

Days

Armc5 mRNA expression in CD4 cells cultured under Th1 and Th17 conditions WT naïve CD4 cells were cultured under Th1 (panel a) or Th17 (panel b) conditions and harvested at 24, 48 and 72 h. Armc5 mRNA expression was measured by RT-qPCR. Experiments were conducted more than 3 times and the normalized ratios of Armc5 versus -actin signals (means ± SEM) of representative experiments are shown. The signal ratios at 0 h are designated as 1.

20

Normalized relative expression (Ratio of ARMC5/ -actin)

Supplementary Figure 14 1.5

1.0

Anti-CD3+anti-CD28+IL-2 Anti-CD3+anti-CD28+IL-2+T GF1 Anti-CD3+anti-CD28+IL-2+ IL-6 Anti-CD3+anti-CD28+IL-2+T GF1+IL-6

0.5

0.0 0

1

2

3

Days

Armc5 mRNA expression in CD4 cells cultured in the presence of different lymphokines WT naïve CD4 cells were cultured in wells coated with anti-CD3 and anti-CD28 (0.5g/ml and 1g/ml during coating) in the presence of IL-2 (2 g/ml). In addition, IL-6 (20ng/ml) or TGF-1 (5 ng/ml), or both was added to culture. The cells were harvested at 24, 48 and 72 h, and their Armc5 mRNA expression was measured by RT-qPCR. Experiments were conducted more than 3 times and the normalized ratios of Armc5 versus -actin signals (means ± SEM) of representative experiments are shown. The signal ratios at 0 h are designated as 1.

21

Supplementary Figure 15

Relative expression (Ratio of ARMC5/ -actin)

CD8+ cells 0.003

p =0.0039

0.002

0.001

0.000

Naive (n=3)

Day8 post-LCMV infection(n=3)

Armc5 mRNA expression in CD8 T cells on day 8 post-LCMV infection CD8 cells were isolated from the spleens of naïve or LCMV-infected (day 8 postinfection) WT mice, with EasySepTM mouse CD8 T-cell isolation kits. Armc5 mRNA levels were measured by RT-qPCR. Means ± SEM of ratios of Armc5 signals versus actin signals from 3 pairs of mice are shown.

22

Supplementary Table 1 Summary of adrenal glands hyperplasia in WT and KO mice Age (months)

Bilateral

Unilateral

Mean ±SD

Hyperplasia

Hyperplasia

WT (n=5)

20 ±1.7

0/5*

1/5*

KO (n=5)

18 ±3.2

3/5*

2/5*

*

number of positive mouse/number of total mice

23