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.
1
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.
2
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.
5
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.
6
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.
7
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.
8
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.
9
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.
11
Supplementary Figure 8
CD25 and CD69 expression in CD4+ and CD8+ cells after anti-CD3 stimulation Spleen cells were stimulated with anti-CD3mAbg/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.
12
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.
13
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.
14
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.
16
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 GF1 Anti-CD3+anti-CD28+IL-2+ IL-6 Anti-CD3+anti-CD28+IL-2+T GF1+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 (0.5g/ml and 1g/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