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Supplementary material

The P4-type ATPase ATP11C is essential for B lymphopoiesis in adult bone marrow

Owen M. Siggs, Carrie N. Arnold, Christoph Huber, Elaine Pirie, Yu Xia, Pei Lin, David

Nemazee & Bruce Beutler

Supplementary Figures 1-5

Supplementary References

Nature Immunology doi:10.1038/ni.2012

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Supplementary Figure Legends

Supplementary Figure 1 A Fas- and Bcl2l11-independent phenotype that cannot be

corrected by a BCL2 transgene. The emptyhive mutation was combined with Faslpr/lpr or

Bcl2l11-/- mutations, or with the EμBCL2-22 transgene, and B cell frequencies were measured

in the blood (a), bone marrow (b, with absolute numbers in c), or spleen (d). ***P > 0.001; ns,

not significant (P > 0.05), as calculated by unpaired t-test comparing wild-type and emptyhive

groups. Each symbol represents an individual mouse from one (b-d) or two (a) independent

experiments.

Supplementary Figure 2 Structural representation of Atp11cemptyhive and Atp11cspelling

mutations. X-ray crystal structure of the Ca2+ ATPase SERCA1 (ref. 1) (PDB: 2EAR, model

generated using MacPyMOL) showing residues corresponding to those mutated in emptyhive

(Q655) and spelling (I355) pedigrees in red. T, transmembrane domain; P, phosphorylation

domain; A, actuator domain; N, nucleotide binding domain.

Supplementary Figure 3 Relative expression of the Atp11c transcript in multiple tissues in

mouse. Atp11c transcript data (gnf1m30854_at probeset) obtained from ref. 2.

Supplementary Figure 4 Surface expression of phosphatidylserine and GM1 ganglioside. (a)

Histograms of Annexin-V and cholera toxin (CTX) staining on the surface of 7-AAD-B220+λ5+

(Annexin-V) and B220+λ5+ (CTX) bone marrow lymphocytes from wild-type (shaded, gray) or

mutant (open, black) mice. (b) Mean fluorescence intensity values for forward scatter (FSC),

Annexin-V and CTX. Data are representative of two experiments, each including three mice

Nature Immunology doi:10.1038/ni.2012

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per genotype. Each symbol represents an individual mouse.

Supplementary Figure 5 Genetic modulation of PtdIns(3,4,5)P3 availability does not affect B

cell deficiency. Mutations known to either increase (a) (Inpp5dstyx, Ptpn6spin) or decrease (b)

(Cd19cre, Cd45belittle) PtdIns(3,4,5)P3 signaling were combined with the emptyhive mutation,

and B cell frequencies were measured in the blood of double mutant mice. Plots are

representative of 1-3 mice per genotype from a single experiment.

Supplementary References

1. Takahashi, M., Kondou, Y. & Toyoshima, C. Interdomain communication in calcium pump as revealed in

the crystal structures with transmembrane inhibitors. Proc Natl Acad Sci USA 104, 5800-5805 (2007).

2. Su, A. I. et al. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci

USA 101, 6062-6067 (2004).

Nature Immunology doi:10.1038/ni.2012

Faslpr/+ Faslpr/lpr100

101

102

A B-D E F

104

105

106

107

A B-D E F

104

105

106

107

A B-D E F

104

105

106

107

A B-D E F

104

105

106

107

Cel

ls

Wild-typeemptyhive

b

Supplementary Figure 1

d

Wild-type emptyhive

IgM

IgD

B220+

CD43

CD

19

B220+

IgM-IgD-

CD23

CD

21

IgM+

Wild-type emptyhive Wild-type emptyhive Wild-type emptyhive

Wild-type emptyhiveWild-type emptyhive

Faslpr/lpr Bcl2l11-/- EμBCL2+

EμBCL2+EμBCL2-

ns

a

c Faslpr/lpr Bcl2l11-/- EμBCL2+

0 102 105103 104

0102

105

103

104

0 102 105103 104

0102

105

103

104

0 102 105103 104

0102

105

103

104

EμBCL2- EμBCL2+104

105

106

107

108

EμBCL2- EμBCL2+104

105

106

107

108

Follicular Marginal zone

Cel

ls

Wild-type emptyhive

CD

19+ (

%)

Bcl2l11+/+ Bcl2l11-/-100

101

102

EμBCL2- EμBCL2+100

101

102

***

ns

Wild-typeemptyhive

32.8

55.311.2

15.2

840.595

14.8

84.6

71.6

22

6.28

92.9

10.6

75.313.3

68.2

27.2

3.82

93.3

56.3

37.6

23.5

74.3

63.3

33.4

19

66.712.7

56.3

36.45.12

61.9

351.43

76.7

12.37.51

26.1

67.75.26

83.4

7.14

15.5

61.6

85.6

0.888

44.8

32.5

Nature Immunology doi:10.1038/ni.2012

Supplementary Figure 2

I355K

Q655*

AP

N

Exoplasm

Cytoplasm

T

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0

2000

4000

6000

8000

1000

0

1200

0

1400

0

1600

0

PancreasSpleen

Fertilized eggOocyteTestisHeart

Skeletal muscleLiver

PlacentaKidney

Large intestineSmall intestine

StomachProstate

DigitsEpidermis

Snout epidermisTongue epidermis

Osteoblasts_Day14Osteoblasts_Day21Osteoblasts_Day5

OsteoclastsMacrophage_1hr LPSMacrophage_7hr LPS

Macrophage_UnstimulatedMast Cells

BoneBone marrow

C2C12Umbilical cord

BladderUterusOvary

B220+ B cellCD4+ T cellCD8+ T cell

Lymph nodeThymus

Embryo day10.5Embryo day6.5Embryo day7.5Embryo day8.5Embryo day9.5

BlastocystsNeuro2ANIH 3T3

M-1Embryonic Stem Feeder Layer

Embryonic Stem No FeederMammary gland.Lact.

Adrenal glandAdipose tissue

Brown fatLung

TracheaMedial olfactory epithelium

PituitaryPreoptic

RetinaAmygdala

CerebellumCerebral cortex

CortexDorsal root ganglion

Dorsal striatumFrontal cortexHippocampus

HypothalamusOlfactory bulb

Spinal cord lowerSpinal cord upperSubstantia nigra

TrigeminalVomeralnasal organ

Septal OrganSeptum (Resp Epith)

Salivary glandThyroid

Relative expression

gnf1m30854_at

Supplementary Figure 3Nature Immunology doi:10.1038/ni.2012

FSC

Annex

in-V

CTX101

102

103 Wild-typeemptyhive

ba

Supplementary Figure 4

Annexin-V CTX

Wild-type emptyhive

B220+λ5+7-AAD-B220+λ5+

100 101 102 103 1040

20

40

60

80

100

% o

f max

mea

n flu

ores

cnce

inte

nsity

Nature Immunology doi:10.1038/ni.2012

a b

Supplementary Figure 5

Atp11c

Thy1.2

CD

19

Thy1.2

CD

19

CD3ε

CD

19

B220

CD

19

Inpp

5dP

tpn6

Ptp

rcC

d19

Atp11c

+/styx

styx/styx

+/cre

cre/cre

+/bel

bel/bel

+/spin

spin/spin

+/Y emp/Y +/Y emp/Y

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

Nature Immunology doi:10.1038/ni.2012