Supplementary information Interaction between … Skokowa1*, Maxim Klimiankou1, , Olga Klimenkova1,...

Supplementary information

Interaction between HCLS1, HAX1 and LEF-1 proteins is

essential for G-CSF-triggered granulopoiesis

Julia Skokowa1*, Maxim Klimiankou1,§, Olga Klimenkova1,§, Dan Lan1, Kshama Gupta1, Kais

Hussein2, Esteban Carrizosa3, Inna Kusnetzova1, Zhixiong Li4, Claudio Sustmann5, Arnold Ganser5,

Cornelia Zeidler1, Hans-Heinrich Kreipe2, Janis Burkhardt3, Rudolf Grosschedl6, Karl Welte1*

1Department of Molecular Hematopoiesis, Hannover Medical School, Hannover, Germany; 2Department of Pathology, Hannover Medical School, Hannover, Germany; 3Department of Pathology

and Laboratory Medicine, Children's Hospital of Philadelphia and University of Pennsylvania School

of Medicine, Philadelphia, USA; 4Institute of Experimental Hematology, Hannover Medical School,

Hannover, Germany; 5Department of Hematology, Hemostasis, Oncology, and Stem Cell

Transplantation, Hannover Medical School, Hannover, Germany; 6Max Planck Institute of

Immunobiology, Department of Cellular and Molecular Immunology, Freiburg, Germany §These

authors contributed equally to this work

e-mail: [email protected]; e-mail:[email protected]

Nature Medicine doi:10.1038/nm.2958

Supplementary Materials and Methods

Sequential chromatin immunoprecipitation (ChIP-re-ChIP). We cross-linked

chromatin from 5 × 107 Jurkat cells by adding 3.7% of formaldehyde to cell culture

medium and incubation for 10 min at the room temperature. The cross-linking

reaction was quenched by adding 100 µl/ml of 1.375 M glycine. Cells were washed

twice with 10 ml of ice-cold PBS, resuspended in 10 ml of ice-cold ChIP cell lysis

buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% Nonidet-40) and incubate for 10 min

on ice. The released nuclei were pelleted by centrifugation for 10 min at 40C. The

supernatant was carefully aspirated. Nuclei were resuspended in 1ml of ice-cold ChIP

nuclei lysis buffer (50 mM Tris-Cl pH 8.0, 10m M EDTA, 1% SDS) supplemented

with protease inhibitor cocktail (Pierce), incubated for 10 min on ice and chromatin

was sonicated. Sonicated chromatin was incubated on ice for 20 min and centrifuged

at high speed for 15 min at 40C. Chromatin was diluted to a final volume of 600

µl/probe with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.2 mM

EDTA, 0,01% SDS, 1.1% Triton X-100) supplemented with protease inhibitor

cocktails. To pre-clear chromatin we added 60 µl of magnetic beads (Dynabeads

Protein G, Invitrogen, cat. Nr. 100.04D) blocked with salmon sperm/BSA and

incubated for 2 h at 40C. For 1st ChIP, we added 7 µg of anti-LEF1 antibody

(monoclonal mouse, cat. Nr. CS200635, Millipore) or isotype control antibody

(normal mouse IgG, cat. Nr. CS200635, Millipore) to the pre-cleared chromatin and

rotated overnight at 40C. After that we added 60 µl of magnetic beads blocked with

salmon sperm/BSA to the chromatin samples and rotated for 2h at 40C. Unbound

chromatin was used as input. Magnetic beads were washed with high-salt ChIP wash

buffer (50 mM HEPES pH 7.9, 500 mM NaCl, 1 mM EDTA, 0,1% SDS, 1% Triton

X-100, 0,1% deoxycholate) followed by washing with 1 ml TE buffer. Each wash was

performed four times by rotation for 10 min at room temperature. Precipitated

DNA:protein complexes were eluted in 100 µl of ChIP elution buffer (50 mM Tris-

HCl, pH 8.0, 10 mM EDTA and 1% SDS) by incubation for 10 min at 680C (1st

ChIP). For re-ChIP (2nd ChIP), DNA-protein complexes from 1st ChIP were incubated

with 7 µg of anti-LEF1, isotype control or anti-HCLS1 antibody (anti-HCLS1, rabbit

polyclonal antibody against full-length native HCLS1 protein, Abnova cat. nr.

H00003059-D01) and 60 µl of pre-blocked magnetic beads. The incubations and

wash steps were performed as described for 1st ChIP. To reverse crosslinks we

incubated samples from 1st ChIP, 2nd ChIP and input overnight at 650C. DNA was


purified using Chromatin IP DNA Purification Kit (Active Motif, cat. Nr. 58002),

according to the manufacturer’s protocol and analyzed by real-time PCR. We

determined the fold changes (enrichment) of target DNA regions (binding sites for

LEF1 protein on the cyclin D1, C/EBPAα and LEF1 gene promoters) in 1st (LEF-1

ChIP) and 2nd (LEF-1 and HCLS1 re-ChIP) samples in comparison to respective

isotype control sample: ∆Ct IP sample = Ct IP sample – Ct input, ∆Ct isotype = Ct

isotype– Ct input isotype; ∆∆Ct= ∆Ct isotype – ∆Ct IP sample for each primer pair.

Final ratio was calculated as F.r.= 1,9^(∆∆Ct), where 1,9 is empirically derived

average of the amplification efficiency for given set of primers and F.r. is a degree of

occupancy of the immunoprecipitated protein at the sequence of interest in the IP

sample with target anibody relative to its level in isotype sample.

To evaluate whether HCLS1 antibody could IP HCLS1 bound to chromatin and are

suitable for ChIP, we performed ChIP with HCLS1 antibody and chromatin from

Jurkat cells. We measured DNA concentration in anti-HCLS1 ChIP DNA sample, in

comparison to anti-LEF-1ChIP DNA sample, isotype ctrl and mock samples. We

could not perform any PCR with anti-HCLS1 ChIP DNA samples, because no

consensus DNA-binding sites for HCLS1 are known. Interestingly, we found that in

anti-HCLS1 ChIP sample concentration of ChIP DNA was significantly higher, in

comparison to isotype control and mock ChIP samples and was comparable to anti-

LEF-1 ChIP DNA sample.

Laser-assisted single cell picking. Participants in this study included: three CN

patients under long-term G-CSF treatment (G-CSF dose ranged between 1.2 and 7.5

mg per kg body weight per day, or once in 2 days); three healthy volunteers during

treatment with G-CSF at a dose of 5 mg per kg body weight per day for 3 days. We

isolated myeloblasts and promyelocytes from bone marrow slides using the PALM

Laser-MicroBeam System (P.A.L.M.) and controlled the purity of individual

populations (100 cells per sample) by qRT-PCR of transcripts encoding myeloid-

specific primary (myeloperoxidase; MPO) and secondary (matrix metalloproteinase-

9; MMP9) granule proteins (data not shown). mRNA was isolated using TRIZOL

reagent (Invitrogen) according to the manufacturer`s protocol with slight

modifications: 10 ng/ml of tRNA and 50 ng/ml of linear polyacrylamide (LPA) (both

Sigma-Aldrich) were added to the TRIZOL.

LEF1, HCLS1 and HAX1 shRNA synthesis, construction of shRNA expression

cassettes and shRNA containing lentiviral vectors. We chemically synthesized


shRNA oligonucleotides corresponding to the human LEF-1, HCLS1, or HAX1 gene

sequences. As control shRNA (ctrl shRNA), we used irrelevant nucleotide of

Arabidopsis thaliana, not mached to any Homo sapiens DNA sequences. All

nucleotides also contained overhang sequences from a 5’ BglII- and a 3’ HindIII-

restriction sites (BioSpring). The oligonucleotide sequences for HCLS1 shRNA has

been published previously1 and for LEF-1 and HAX1 shRNA are available on

request. We inserted the oligonucleotides into the BglII/HindIII-digested pSUPER-

derived plasmid to generate pSUPER-LEF-1, pSUPER-HCLS1 or pSUPER-HAX1

and verified the isolated clone by DNA sequencing. To generate lentiviral transgenic

plasmids containing shRNA expression cassettes located in the U3 region of the ∆∆∆∆3´-

LTR we used pRRL.PPT.SF.DsRedEx.pre, which is a derivative of the standard

lentiviral vector pRRL.PPT.PGK. DsRedEx.pre (kindly provided by Luigi Naldini,

Milano, Italy) using an internal SFFV promoter2. To generate the lentiviral LEF-1,

HCLS1 and HAX1 shRNA plasmids, we digested the pSUPER-LEF-1, pSUPER-

HCLS1 and pSUPER-HAX1 with SmaI and HindII and ligated the resulting DNA

fragments (360 bp) into the SnaBI site of the pRRL.PPT.SF.DsRedEx.pre. The

lentiviral plasmid encodes RFPEXPRESS as reporter gene.

HCLS1 cDNA synthesis and construction of HCLS1 cDNA and HCLS1_NLS or

HCLS1_Y397F cDNA containing lentiviral vectors We amplified 1,560 bp HCLS1

cDNA and cloned it into pRRL.PPT.SF.i2GFPpre vector. This vector is a derivative

of the standard lentiviral vector pRRL.PPT.PGK.GFPpre (kindly provided by Luigi

Naldini, Milano, Italy)2. Details are available on request. To construct the

HCLS1_NLS, we cut pRRL.PPT.SF.HCLS1.i2GFPpre with BamHI, treated it with

Klenow polymerase, redigested with SacI and ligated with a StuI/SacI fragment

(containing the HCLS1 NLS IRES GFP cassette) of the same vector. We introduced

mutation in Tyrosine 397 of pRRL.PPT.SF.HCLS1.i2GFPpre by replacement of Tyr

397 with alanine.

Gene expression microarray analysis. CD34+ cells were transduced with HCLS1

shRNA or ctrl shRNA RFP lentiviral constructs, RFP+ cells were isolated on day 4 of


transduction. RNA was isolated using RNA Isolation Kit from Qiagen. RNA was

subjected to microarray analysis using Affymetrix Microarray Platform. The

Genechip WT cDNA Synthesis and Amplification Kit was used to make double-

stranded cDNA from total RNA, which was then labeled with biotin (Genechip WT

Terminal Labeling Kit). After chemical fragmentation of the biotin-labeled cRNA

targets, they were hybridized to Affymetrix Human Genom WT microarrays using the

Fluidics Station 450 and scanned using the Affymetrix Genechip Scanner 3000 with

Genechip Operating Software 1.4 (Affymetrix, Santa Clara, CA). Data analysis was

performed using Affymetrix Expression Console Version 1.1 for invariant set

normalization and PARTEK (www.partek.com) software was used for identification

of differentially expressed genes.

Tissue array expression analysis in clinical bone marrow samples. Protein

expression was analysed in clinical samples from 52 patients (supplementary Table

4). Formalin-fixed and paraffin-embedded (FFPE) bone marrow biopsies and lymph

node resection in all cases, including controls, had been taken as part of standard

clinical care for the evaluation of bone marrow and lymph node status for exclusion

of an underlying neoplasm. After the diagnosis had been established (Institute of

Pathology, Hannover Medical School), the archived tissue was considered as

remained material, unnecessary for patient’s treatment. The retrospective analysis of

these samples was approved by the local Ethics Committee, Hannover Medical

School. Representative areas were selected for generation of a FFPE tissue array with

samples from 38 patients with acute and chronic stage myeloid neoplasms, 8 non-

neoplastic controls and 1 negative control (canine cardiomyocytes). Myeloid

malignancies comprised AML M0 (n = 9), AML M1 (n = 8), AML M2 (n = 8), AML

M4 (n = 12), AML M5 (n = 9), AML M6 (n = 2) and post-myelodysplastic syndrome

(MDS) AML (n = 4) (Suppl. Table 3). Controls comprised normal medullar

haematopoiesis without maturation defects (n = 5 cases/6 biopsies; in one case bone

marrow biopsies from right and left crista iliaca had been taken for exclusion of

Ewing sarcoma infiltrates and both samples were analysed for intraindividual control)

and abdominal, cervical and lung lymph node samples (n = 3 cases/4 samples; in one

case two areas of an abdominal lymph node were analysed). Four control bone

marrows were placed at the corners and one control bone marrow and one lymph

node in the centre of the micro array; all other cases were randomly organised on the


array. Immunohistochemistry was performed with a ZytoChem-Plus HRP Polymer kit

(Zytomed Systems, Berlin, Germany). Xylol-deparaffinised and re-hydrated FFPE-

sections (~3 µm) were pre-treated in an autoclave, incubation with peroxide (3% in

70% ethanol) and than blocking solution prior to over night-incubation in a humified

camber at 4°C with anti-HS1 monoclonal mouse antibody (1:25, BD Biosciences

Pharmingen, Franklin Lakes, NJ, USA). After visualization of immunostaining with

DAB sections were counterstained with haematoxylin. Analysis was performed in

duplicate and evaluation was performed separately according to the following scoring

system: - = no staining; + = weak staining; ++/+++ = strong staining; ++++ = very

strong staining/lymph node (pos. control).

Vitamin B3 treatment. We treated six healthy individuals with Vitamin B3

(Nicotinsäureamid, JENAPHARM, Germany; Zul.-Nr.: 3000282.00.00) doses 10

mg/kg/day for 7 days. Three persons were treated twice in two independent

experiments. Informed consent was obtained from all subjects. We obtained approval

for this study from the Hannover Medical School’s institutional review board.

Supplementary references

1. Gomez TS, McCarney SD, Carrizosa E, Labno CM, Comiskey EO, et al. HS1

functions as an essential actin-regulatory adaptor protein at the immune

synapse. Immunity. 24, 741-52 (2006).

2. Dull, T. et al. A third-generation lentivirus vector with a conditional

packaging system. J. Virol. 72, 8463-8471 (1998).


Supplementary figures


a

LEF-1-HA Tag

HAX1

IP: HA TagWB: HA Tag

IP: HA TagWB: HAX1

Supplementary Figure 1


b

WB:HCLS1

inputG-CSF 0` 30`

WB:Syk

WB:Lyn

IP: HCLS1

HCLS1

Syk

* * Lyn

phHCLS1

G-CSF 0` 30`

ß-actin

a

Lyn

HCLS1

input

0` 30` G-CSF



b ctrlG-CSF

a

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m

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0

200

400

600

CD33+ cells

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*

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G-CSF - + - + + +

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a

b

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c- G-CSF+ G-CSF*


a

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total HCLS1 / phospho-HCLS1

total HCLS1Isotype ctrl

phospho-HCLS1

b

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cctrl shRNAanti-HAX1a shRNAanti-HAX1b shRNA

100

80

60

40

20

0100 101 102 103 104

FL2-H

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0100 101 102 103 104 100 101 102 103 104

FL2-HFL2-H


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ratio

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*

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ratio

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a

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b


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100

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num

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+ IL-3, SCF, TPO, GM-CSF, G-CSF

mockctrl shRNAanti-HCLS1b shRNA

anti-HAX1a shRNAanti-HCLS1f shRNA

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CFU-G CFU-M

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* * * ** * * *

ponceau ponceau


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ctrl

sh

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shR

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0.26 % 0.75 %

0.15 % 0.02 %

0.08 % 0.06 %

0.12 % 0.03 %

0.65 % 63.2 %

0.01 % 19.4 %

0.03 % 14.5 %

0.02 % 17.9 %

a

100

101

102

103

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100 101 102 103 104

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100 101 102 103 104

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FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height

100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height

100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height


isotype ctrl CD11b isotype ctrl CD11b

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0.38 % 0.12 % 0.05 % 22.3 %

b

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100

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100

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100

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100

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FL2

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100

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100

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100

101

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103

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100

101

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103

104

FL2

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100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height

100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height100 101 102 103 104

FL1-H: FL1-Height


c

0

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x xxx x

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*

C/EBPα promoterx xx

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a

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*

*

c

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*

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100

150

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ter


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a

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b

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NA

ratio

, AU

0

25

50

75

ctrl shRNAanti-LEF-1a shRNA

*

HCLS1

d

anti-LEF-1b shRNA

*

0.3 *

0.1

0.2

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0.2

0.3

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oter

(RLU

)

MU

T H

CLS

1 pr

omot

er(R

LU)

LEF-1 b. s. 1 LEF-1 b. s. 2

ChIP

neg. ctrl for ChIP

0

0.01

0.02

0.03

LEF1 +–

*

f - G-CSF+ G-CSF

–

*

HCLS1 HCLS1x x

LEF1 binding sites Mut. LEF1 binding sitesx

0

0.01

0.02

0.03

LEF1 + ––LEF-1 shRNA + +

– ––––

+

*

–

HC

LS1

prom

oter

(RLU

)

HC

LS1

prom

oter

(RLU

)

LEF10

+ –0

LEF1 + –

HCLS1 HCLS1x x

LEF1 binding sites Mut. LEF1 binding sitesx

HC

LS11

pro

mot

er

MU

T H

CLS

1 pr

omot

er



a

0` 15`G-CSF 30`0

*25

50

75

pAkt

ser

473/

tota

l Akt

ratio

, %

*

0` 15` 30`

100

0` 15`G-CSF 30`0

*

25

50

75

pPI3

K p

85/to

tal P

I3K

p85

ratio

, %

*

0` 15` 30`

ctrlCN100

ctrlCN

b



2` 5`+ G-CSF 3`

- G-CSF+ G-CSF

23.4% 9.85%11.9%

b

HAX1 shRNActrl shRNAHCLS1 shRNA

ctrl shRNA

53.5%34.9%

F-actin F-actin

a

100 100 100

100

80

60

40

20

0

% o

f Max

100 101 102 103 104

FL3-H

100

80

60

40

20

0

% o

f Max

100101 102 103 104

FL3-H


ctrl

shR

NA

HC

LS1

shR

NA

HA

X1

shR

NA

23.4% 9.85%11.9%

F-actin

80

60

40

20

0100 101 102 103 104

FL3-H

80

6040

20

0100 101 102 103 104

FL3-H

8060

40

20

0100 101 102 103 104

FL3-H100

80

60

40

20

0100 101 102 103 104

FL3-H

10080

6040

20

0100 101 102 103 104

FL3-H

10080

60

40

20

0100 101 102 103 104

FL3-H100

80

60

40

20

0100 101 102 103 104

FL3-H

100

80

60

40

20

0100 101 102 103 104

FL3-H

100

80

60

40

20

0100 101 102 103 104

FL3-H


a

Lin-/c-Kit+/Sca1-

GMP

CMPMEP

CD34

Fc γγ γγ

RII/

III

Lin-/c-Kit+/Sca1-


WTHCLS1-/-

GMP

CMP

MEPGMP

CMP

MEP

CD34

Fc γγ γγ

RII/

III

24.5 %

51.2 %19 %

18 %

62 %17.3 %

FL2

-H

100

101

102

103

104

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104

FL2

-H

FL3-H FL3-H

WT3 cd34 fcg…FSC-H, SSC-H subset HS! 2 cd34 fcg…FSC-H, SSC-H subset


a

0

40

80

120

WTHCLS1-/-

CFU-GM BFU-E

+ IL-3, SCF, TPO, GM-CSF, G-CSF

CFU-G CFU-M CFU-Mega

CF

Us

num

ber

*

**


b

targ

et g

ene/

ß-a

ctin

mR

NA

ratio

, AU

0

5

10

15

C/EBPα

WTHCLS1-/-

0

10

20

*

LEF-1

*

C/EBPβ0

40

80

0

20

40

60

0

5

10

Nampt SIRT1

*


b mockctrl shRNA

anti-LEF1a shRNA

anti-HCLS1b shRNAanti-HCLS1f shRNA

a

HC

LS1/

ß-a

ctin

mR

NA

ratio

, AU

ctrl CD33+ cells

0

100

200

400 *

300

AML

*

ctrl CD34+ cells


targ

et g

ene/

ß-a

ctin

mR

NA

ratio

, AU

0

5

10

15

LEF-1

0

100

200

300

survivin

anti-LEF1a shRNA

0

50

100

150

cyclin D1

**

**

**

targ

et g

ene/

ß-a

ctin

mR

NA

ratio

, AU

0

20

40

60

HCLS1

***

**

**

*

*

*

anti-LEF1b shRNA


Supplementary Figure Legends

Supplementary Figure 1. Overexpressed LEF-1 protein interacts with

endogenous HAX1 protein in 293T HEK cells

(a) HEK293T cells were transfected with pcDNA-HCLS1 and pcDNA-LEF-1-HA-

Tag (LEF-1-HA WT). 36 hours after transfection, cells were lysed and LEF-1 protein

was immunoprecipitated with protein G agarose beads conjugated with anti-HA-Tag

antibody. Co-immunoprecipitation of overexpressed LEF-1 and endogenous HAX1

proteins was detected by WB. Representative WB image is depicted.

Supplementary Figure 2. HCLS1 co-immunoprecipitated with Lyn and Syk in

bone marrow CD34+ cells

(a) We treated CD34+ bone marrow cells from two healthy individuals (ctrl) with 10

ng/ml of G-CSF, at indicated time points, we lysed cells in Laemmli buffer and

analysed phospho-HCLS1 protein and ß-actin expression by WB. Representative WB

image of phHCLS1 protein and ß-actin of cells of one healthy individual.

(b) CD34+ cells were treated or not with 10 ng/ml of G-CSF; endogenous HCLS1

protein was immunoprecipitated from cell lysates with rabbit polyclonal anti-HCLS1

antibody; interaction with endogenous Lyn and Syk proteins was detected by Western

blot (WB) analysis with mouse monoclonal anti-Lyn and mouse monoclonal anti-Syk

antibody; upper panel: representative WB image is depicted, isotype control was

negative and therefore is not presented. Lower panel: Representative image of Lyn

and HCLS1 inputs for IP are presented.

Supplementary Figure 3. Nicotinamide (NA, vitamin B3) and Nampt induce

HCLS1 mRNA expression in hematopoietic cells

(a) We treated 3 healthy individuals with Vitamin B3 (nicotinamide, NA) (10

mg/kg/day) for one week and measured mRNA expression of HCLS1 in bone marrow

CD34+ and CD33+ cells quantified by qRT-PCR and normalized to ß-actin on day 3

of Vitamin B3 treatment, data represent means ± s.d. of triplicates, *, P < 0.05. (b)

We treated CD34+ cells of healthy individuals (n = 3) with 10 ng/ml of G-CSF or 10

ng/ml of Nampt for 24 hours. HCLS1 mRNA expression is normalized to ß-actin and

is presented as arbitrary units (AU). Data represent means ± s.d. and are derived from

three independent experiments each in triplicates (*, P < 0.05).


Supplementary Figure 4. Diminished HCLS1 and unaffected HAX1 mRNA

expression in myeloid cells of CN patients

(a) HAX1 mRNA expression in CD33+ cells of studied groups: eight CN patients

harbouring HAX1 mutations, three patients with neutropenia associated with

congenital disorders of metabolism (MN) and three healthy G-CSF-treated individuals

(ctrl), HAX1 mRNA expression is normalized to ß-actin and is presented as arbitrary

units (AU), data represent means ± s.d. of triplicates. (b) We isolated myeloblasts and

promyelocytes from bone marrow slides of G-CSF treated CN patients harbouring

HAX1 mutations (n = 3) and G-CSF treated healthy individuals (n = 3) using the

PALM Laser-MicroBeam System. For qRT-PCR we isolated RNA using TRIZOL

reagent (Invitrogen), amplified cDNA using random hexamer primer (Fermentas) and

measured HCLS1 and HAX1 mRNA expression using SYBR green qPCR kit

(Qiagen). Target gene mRNA expression was normalized to ß-actin and was

represented as arbitrary units (AU). Data represent means ± s.d. and are derived from

triplicates, *, P < 0.05. (c) LEF-1 mRNA expression in CD33+ cells of studied

groups: eight CN patients harbouring HAX1 mutations, three patients with

neutropenia associated with congenital disorders of metabolism (MN) and three

healthy G-CSF-treated individuals (ctrl), LEF-1 mRNA expression is normalized to ß-

actin and is presented as arbitrary units (AU), data represent means ± s.d. of triplicates

(*, P < 0.05).

Supplementary Figure 5. Defective phosphorylation of HCLS1 in response to G-

CSF in CD 33+ cells of CN patients and in CD34+ cells of healthy individuals

transduced with HAX1 shRNA

(a) We treated CD33+ bone marrow cells of two healthy individuals (ctrl) and two CN

patients (CN) with 10 ng/ml of G-CSF, at indicated time points, we harvested, fixed,

permeabilized cells and quantified amounts of intracellular total- and phospho-

HCLS1 protein using immunostaining and FACS analysis. Representative histogram

is depicted. (b-d) We transduced CD34+ cells of healthy individuals (n = 3) with ctrl-

RFP shRNA or two different anti-HAX1-RFP shRNAs, sorted RFP+ cells on day 4 of

culture, we measured: (b) HAX1 mRNA expression levels by real-time qRT-PCR

(HAX1 mRNA expression is normalized to ß-actin and is presented as arbitrary units

(AU). Data represent means ± s.d. and are derived from three independent

experiments each in triplicates (*, P < 0.05) and (c) HAX1 protein levels by WB


(representative WB image is depicted); (d) We treated sorted cells with 10 ng/ml of

G-CSF, at indicated time points, we harvested, fixed, permeabilized cells and

quantified amounts of cells positive for intracellular total- and phospho-HCLS1

protein using immunostaining and FACS analysis. Percentage of phosphoHCLS1

positive cells within population of total HCLS1 protein positive cells is represented.

Data represent means ± s.d. and are derived from three independent experiments each

in duplicate (*, P < 0.05).

Supplementary Figure 6. HCLS1 is required for LEF-1-target genes mRNA

expression, but not for mRNA expression of components of Nampt signaling

(a, b) We transduced CD34+ cells from healthy individuals (n = 3) with two different

constructs of anti-HCLS1 shRNA, or ctrl shRNA contained RFP, sorted RFP+ cells,

treated sorted cells with 10 ng/ml of G-CSF for 12 hours and measured mRNA

expression of indicated genes using qRT-PCR. mRNA expression is normalized to ß-

actin and is presented as arbitrary units (AU). Data represent means ± s.d. and are

derived from three independent experiments each in triplicate, *, P < 0.05. Effects of

HCLS1 shRNA on HCLS1 inhibition are represented in Fig. 2d.

Supplementary Figure 7. HAX1 is required for LEF-1-target genes mRNA

expression

(a) We transduced CD34+ cells from healthy individuals (n = 3) with two different

constructs of anti-HAX1 shRNA or ctrl shRNA contained RFP, sorted RFP+ cells,

treated sorted cells with 10 ng/ml of G-CSF for 12 hours and measured mRNA

expression of indicated genes using qRT-PCR. mRNA expression is normalized to ß-

actin and is presented as arbitrary units (AU). Data represent means ± s.d. and are

derived from three independent experiments each in triplicate, *, P < 0.05. Effects of

HAX1 shRNA on HAX1 inhibition are represented in Suppl. Fig. 4b,c.

Supplementary Figure 8. HCLS1 and HAX1 are involved in myeloid

differentiation in vitro

We transduced CD34+ cells from healthy individuals (n = 3) with two different anti-

HCLS1 shRNAs or two different anti-HAX1 shRNAs and ctrl shRNA constructs

contained RFP and sorted RFP+ cells. (a) HCLS1 and HAX1 mRNA expression

levels were measured by real-time qRT-PCR (data represent means ± s.d. and are


derived from three independent experiments each in duplicate, *, P < 0.05) and (b)

HCLS1 and HAX1 protein levels were assessed by WB (representative WB image is

depicted). (c) We performed CFUs assay of transduced and sorted cells, as described

in Materials and Methods. Data represent means ± s.d. and are derived from two

independent experiments each in duplicate, *, P < 0.05.

Supplementary Figure 9. HCLS1 and HAX1 are required for ATRA-triggered

myeloid differentiation of the promyelocytic cell line NB4

We transduced the promyelocytic cell line NB4 with two different anti-HCLS1

shRNAs or two different anti-HAX1 shRNAs and ctrl shRNA constructs contained

RFP and sorted RFP+ cells and treated or not with 0.1 mM of ATRA. On day 2 of

stimulation, we measured CD11b surface expression in RFP+ cells using FACS.

Representative FACS images are depicted in (a), % of CD11b+ cells is shown in (b).

Data represent means ± s.d. and are derived from two independent experiments each

in duplicate (*, P < 0.05). (c) mRNA expression levels of HCLS1 and HAX1 in

transduced and sorted NB4 cells, were measured by real-time qRT-PCR (data

represent means ± s.d. and are derived from three independent experiments each in

triplicate, *, P < 0.05).

Supplementary Figure 10. LEF-1-, HCLS1- and HAX1- dependent activation of

the LEF-1 gene promoter

(a) We measured the effects of LEF-1 on G-CSF- dependent activation of the LEF-1

gene promoter in CD34+ cells of healthy individuals (n = 3) transfected with cDNA of

LEF-1 cDNA in the presence or absence of HCLS1-, or HAX1 shRNA. Data


triplicate, *, P < 0.05. (b) We measured the effects of HCLS1, HAX1 and LEF-1 on

G-CSF- dependent activation of the LEF-1 gene promoter in CD34+ cells of healthy

individuals (n = 3) transfected with cDNA of HCLS1 in the presence or absence of

LEF1-, or HAX1 shRNA. Performance of the reporter gene assay is described in

Materials and Methods. Data represent means ± s.d. and are derived from three

independent experiments each in triplicate, *, P < 0.05. (c) We measured the effects

of HCLS1 and LEF-1 on G-CSF-dependent activation of LEF-1 gene promoter with

mutated LEF-1 binding sites in CD34+ cells of healthy individuals (n = 3) transfected

with LEF-1 cDNA, or HCLS1 cDNA, or combination of both. Data represent means


± s.d. and are derived from three independent experiments each in triplicate.

Schematic presentation of promoter construct with five mutated LEF-1 binding sites

(pink cycles). Performance of the reporter gene assay is described in Materials and

Methods.

Supplementary Figure 11. LEF-1- and HCLS1-dependent activation of C/EBPαααα

gene promoter

(a) We measured the effects of HCLS1 on G-CSF- and LEF-1/ß-catenin- dependent

activation of C/EBPα gene promoter in CD34+ cells of healthy individuals (n = 3)

transfected with HCLS1 cDNA in combination with LEF-1 cDNA (LEF-1), or LEF-1

with mutated HCLS1 binding site (LEF-1_HCLS1_bs_MUT) or dominant negative

LEF-1 (dnLEF-1). Data represent means ± s.d. and are derived from three

independent experiments each in triplicate, *, P < 0.05. (b) We assessed the effects of

HCLS1 on C/EBPα gene promoter activation after inhibition of LEF-1 or HAX1

using specific anti-LEF-1 or anti-HAX1 shRNA constructs. Data represent means ±

s.d. and are derived from three independent experiments each in triplicate, *, P <

0.05. (c) We analysed the effects of LEF-1 on C/EBPα gene promoter activation after

inhibition of HCLS1 or HAX1 using specific anti-HCLS1 or anti-HAX1 shRNA

constructs. Data represent means ± s.d. and are derived from three independent

experiments each in triplicate, *, P < 0.05. (d) We measured the effects of HCLS1 on

G-CSF-dependent activation of C/EBPα gene promoter with mutated LEF-1 binding

sites in CD34+ cells of healthy individuals (n = 3) transfected with LEF-1 cDNA in

combination with HCLS1 cDNA. Data represent means ± s.d. and are derived from

three independent experiments each in triplicate. On the left side of the Figures (a)

and (c) are schematic presentations of C/EBPα gene promoter construct with LEF-1

binding sites (pink cycles). Performance of the reporter gene assay is described in

Materials and Methods.

Supplementary Figure 12. LEF-1- and HCLS1-dependent activation of the LEF-

1 gene promoter as well as of C/EBPαααα mRNA expression

We measured the effects of HCLS1 on LEF-1- and dnLEF-1- dependent activation of

the LEF-1 reporter gene (a,b), or TOP reporter (c) in HEK 293 cells, as described in

Materials and Methods. Data represent means ± s.d. and are derived from three


independent experiments each in triplicate, *, P < 0.05; bottom lines: schematic

representation of promoter constructs, TOP promoter contains six and LEF-1

promoter five LEF-1/TCF binding sites (pink cycles). (d) We transduced CD34+ cells

of healthy individuals (n = 3) with two different anti-LEF-1 shRNAs in combination

with LEF-1 cDNA, or dnLEF-1 cDNA, or LEF-1_HCLS1_MUT cDNA, sorted RFP+

cells on day 4 of culture and measured C/EBPα mRNA expression using qRT-PCR.

mRNA expression is normalized to ß-actin and is presented as arbitrary units (AU).


in triplicate, *, P < 0.05.

Supplementary Figure 13. Feed-back regulation of HCLS1 by LEF-1

(a, b) We transduced CD34+ cells of healthy individuals (n = 3) with anti-LEF-1-RFP

shRNA, or ctrl-RFP shRNA, sorted RFP+ cells and assessed: (a) HCLS1 mRNA

expression levels by real-time qRT-PCR, data represent means ± s.d. and are derived

from three independent experiments each in triplicate, *, P < 0.05; (b) HCLS1 and

LEF-1 protein levels by WB, representative WB images are presented; (c) putative

LEF-1 binding sites (depicted in pink) and C/EBPa binding sites (depicted in blue)

were mapped on the 5400 bp HCLS1 promoter, using Genomatix Software; (d)

chromatin immunoprecipitation (ChIP) assay of nuclear extracts of CD33+ bone

marrow cells using rabbit polyclonal anti-LEF-1 antibody; PCR products were

amplified using primer pairs flanking two LEF1 binding sites (LEF-1 b.s. 1 and LEF-

1 b.s. 2) of the HCLS1 gene promoter and a primer pair for negative control of ChIP

reaction (neg. ctrl for ChIP), as described in Materials and Methods. No Ab, no

antibody; isotype, isotype antibody control; anti-LEF-1, IP with anti-LEF-1 antibody;

(e) We measured the effects of LEF-1 on activation of the HCLS1 reporter gene

constructs in HEK 293 cells transfected or not with LEF-1 cDNA. Performance of the

reporter gene assay is described in Materials and Methods. Data represent means ±

s.d. and are derived from three independent experiments each in triplicate, *, P <

0.05; Schematic presentation of promoter construct with two LEF-1 binding sites

(pink cycles); (f) We measured the effects of LEF-1 on G-CSF-dependent activation

of HCLS1 gene promoter in CD34+ cells of healthy individuals (n = 3) transfected or

not with LEF-1 cDNA, or with LEF1 shRNA. Performance of the reporter gene assay


is described in Materials and Methods. Data represent means ± s.d. and are derived

from three independent experiments each in triplicate, *, P < 0.05.

Supplementary Figure 14. Severely diminished levels of phospho-PI3K p85 (Tyr

458) and of phospho-Akt (Ser 473) in CD34+ cells of CN patients, as compared to

healthy individuals.

We treated or not CD34+ cells of healthy individuals (n = 3) or of CN patients (n = 3)

with 10 ng/ml of G-CSF. At indicated time points, we harvested, fixed, permeabilized

cells and quantified amounts of (a) intracellular total PI3K p85 and phospho-PI3K

p85 (Tyr 458) and of (b) total Akt and phospho-Akt (Ser473) using immunostaining

and FACS analysis. Percentage of phospho- to total protein positive cells is

represented. Data represent means ± s.d. and are derived from two independent

experiments each in duplicate (*, P < 0.05).

Supplementary Figure 15. HCLS1 and HAX1 control F-actin rearrangement in

response to G-CSF

(a) We transduced CD34+ cells of healthy individuals (n = 3) with anti-HCLS1-RFP

shRNA, anti-HAX1-RFP shRNA or ctrl-RFP shRNA, sorted RFP+ cells, subsequently

fixed, permeabilized and stained RFP+ cells with phalloidin for one hour at 4 °C and

assessed intracellular F-actin amount in RFP+ cells by FACS. Representative

histograms showing F-actin staining are depicted; (b) We treated transduced and

sorted HCLS1-RFP+, HAX1-RFP+ or ctrl-RFP+ cells with 10 ng/ml of G-CSF for

indicated time points, subsequently fixed, permeabilized and stained cells with

phalloidin for one hour at 4 °C and assessed intracellular F-actin amount in RFP+ cells

by FACS. Representative histograms showing F-actin staining are depicted.

Supplementary Figure 16. FACS analysis of myeloid cells in bone marrow of

HCLS1-/- and WT mice

We harvested bone marrow cells from the long bones of WT (n = 4) and HCLS1-/- (n

= 4) mice using ice-cold PBS. After subsequent Ficoll gradient centrifugation, cells

were stained with appropriate antibodies and analysed by FACS. Representative

FACS images of the myeloid compartment in HCLS1-/- and WT mice: we gaited

lineage-negative (neg for CD3, CD4, CD8, CD19, B220, Gr-1), c-kit positive, sca-1

negative (LSK) bone marrow cells and assessed FcgRII/III and CD34 expression


within LSK cells. Upper gate: CD34+/FcgRII/IIIhi granulocyte/macrophage

progenitors (GMP); middle gate: CD34+/FcgRII/IIIlow common myeloid progenitors

(CMP); bottom gate: CD34-/FcgRII/III- megakaryocyte/erythrocyte progenitors

(MEP). Percentage of total bone marrow cells is presented.

Supplementary Figure 17. Defective LEF-1-dependent granulopoiesis in HCLS1-

/- mice

(a) We performed CFUs assay using bone marrow of HCLS1-/- (n = 4) and WT mice

(n = 4) as described in materials and methods. Data represent means ± s.d. and are

derived from three independent experiments each in duplicate, *, P < 0.05, **, P <

0.01. (b) We isolated mRNA from CFU-G cells of of HCLS1-/- (n = 3) and WT (n =

3) mice and measured mRNA expression of indicated genes by real-time PCR;

mRNA expression is normalized to ß-actin and is presented as arbitrary units (AU).


in triplicate, *, P < 0.05.

Supplementary Figure 18. HCLS1 mRNA expression is dramatically elevated in

primary blasts of AML patients and effects of the inhibition of HCLS1 or LEF-1

on LEF-1 target genes mRNA expression in primary blasts of AML patients

(a) We measured HCLS1 mRNA expression levels in primary blasts of 10 patients

with AML by real-time qRT-PCR. mRNA expression is normalized to ß-actin and is

presented as arbitrary units (AU). Data represent means ± s.d. and are derived from

three independent experiments each in triplicate, *, P < 0.05. (b) We transduced blasts

of patients with AML (n = 3) with two different constructs of anti-HCLS1 shRNA, or

anti-LEF-1 shRNA or ctrl shRNA contained RFP, sorted RFP+ cells and measured

mRNA expression levels of indicated genes by real-time qRT-PCR. mRNA

expression is normalized to ß-actin and is presented as arbitrary units (AU). Data


triplicate, *, P < 0.05.


Supplementary tables 2, 3, 4, 5


Supplementary Table 2. Peripheral blood counts of WT and HCLS1-/- mice.

Leukoc. Neutroph. Eosinoph. Lymphoc. Monoc. Erythroc. Hb Hct MCV Thromboc.

WT 5670

±2077.75 1194.5 ±282.61

13.5 ±23.12

4240.5 ±789.69

221.5 ±337.78

9.065 ±0.59

12.55 ±1.67

39.35 ±2.84

43.35 ±1.46

827 ±332.63

HCLS1-/- 5340 ±1206.83

720,12 ±60.20

35.5 ±60.20

4130.5 ±1950.31

453.5 ±197.47

9.275 ±0.71

13.05 ±1.90

40.4 ±2.77

43.85 ±1.53

665 ±486.72

ttest 0.49380687 0.035 0.2498141 0.1677626 0.1851591 0.5324102 0.5528916 0.4022995 0.626326 0.7838714


Supplementary Table 3.AML patients characteristics and intensity of HCLS1 expression in tissue microarrays

Diagnosis Karyotype Gender Age Bone marrow blasts, %

Intensity of HCLS1 protein staining in

BM blasts

M0 46,XX,t(1;11)(q25;q23),-7,+r 13/46,XX 2 F 58 95 +++

M0 47,XX,+13 2/46,XX 14. F 57 95 +++

M0 92,XXX,-X,+13 5/50,XX,+X,+4,+8,+21 2/46,XX 14. F 61 80 +++

M0 46,XX [25]. F 44 90 ++

post-MDS AML M0 46,XX,t(4;12)(q11;p13) [22]. F 39 80 ++

M0 46,XX [25]. F 34 80 +++

M0 unknown unkn. unkn. <5 ++

M0 44,XY, del(5)(q15),-7,-16,13/46,XY M 69 unknown ++

M0 unknown F 44 unknown +++

M1 48-49,XX,+6,+8,der9,-21,+mar1-4 [4]/46,XX[13]. F 54 90 +++

M1 46,XY, t(1;7)(p34;q33), t(8;21)(q22;q22) 10/46,XY 7. M 74 80 ++

M1 45,XX,t(3;4)(q26;q2?6),-7,inv(9)(p11q13)c 15. F 56 90 +++

M1 46,XY[15]. M 72 80 ++


M1 47,XY,+9 13/46,XY 2. M 70 50 +++

M1 46,XX F 50 unknown +++

M1 46,XY M 48 unknown +++

M1 46,X,t(X;3)(q25?,p11), 2/46,X,idem…. F 30 unknown ++

M2 43,XY,-3,der(5)add(5)(p12)t(5;5)(p12;q33),add(6)(p23),-7,?del(11) (q23),-20, dic(20;21)(q11;p12) [18]/46,XY [2]. M 62 unknown -

M2 46,XY [25]. M 64 70 ++

M2 45,XY,-13 4/46,XY 20. M 75 35 +

M2 46,XY,t(10;11)(p13;q14 21) 11/46,XY 5. M 19 90 +++





M4 46,XY M 55 unknown ++

M4 unknown M 46 >20 ++

M4 46,XY,del(11)(p11p13),inv(16)(p13q22) 25/47,XY,idem,+8 2. M 56 95 +++

M4eo unknown M 67 55 +


M4eo unknown M 66 30 ++

M4eo 46,XY,inv(16)(p13q22),+22 18/46,idem,idic(22)(p11),-22 2/46,idem,-22 1 M 62 >20 +

M4eo 46,XY,inv(16)(p13q22) 14/46,XY 11 M 32 90 ++

M4 46,XX,der(2)t(2;11)(q22;q14)?add(11)(q24),t(16;16)(p13;q22) [15] F 23 90 +++

M4 48,XY, add(5)(q14), ?t(6;11)(q27;q23), del(16)(p11), +21, M 44 unknown +++

M4 45,XY, -7, Monosomie 7 M 51 unknown +++


M4 46,XX F 58 unknown ++

M5 46,XX [20]. F 51 95 +++


M5a unknown unkn. unkn. unknown +

M5a 46,XX [25]. F 61 90 ++

M5b 46,XX [25]. F 69 70 +++

M5b 46,XX [16]. F 66 55 +++

M5b 47,XX,+8 1/46,XX 19 F 74 90 +++

M5b 46,XX F 75 unknown +++


M5a 45,XX, -7 F 61 unknown +++

M6a unknown M 55 >20 +

M6a unknown M 60 >20 +++

post-MDS AML 41-48XY aberrant 3, -5,-6,+11,-12,-13 aberrant 14, +1 M 57 unknown ++

post-MDS AML 45-46,Xydel(3),-5 aberrant 7,9,-13,21,+21,t(8;11) M 74 unknown ++

post-MDS AML 46,XY M 77 unknown ++

post-MDS AML 44,X,-Y,add(2)(q34),-12 M 62 unknown +++


Supplementary Table 4.Expression of cytokines and cytokine receptors in AML blasts from public databases

Data set 1 GSE1729http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1729

GeneSymbol probeset_ID param Fval p-val p-val.adjCSF3 207442_at Karyotype 6.740835689 1.65E-05 9.31E-05MPL 207550_at Karyotype 15.75020219 5.48E-09 4.25E-08EPOR 209962_at Karyotype 3.212466038 0.004384945 0.016991663EPOR 209963_s_at Karyotype 2.418211588 0.022731502 0.07417648MPL 216825_s_at Karyotype 2.7192153 0.011956917 0.041184936EPOR 37986_at Karyotype 3.781446581 0.001490804 0.007702486


GeneSymbol probeset_ID param Fval p-val p-val.adjCSF3R 203591_s_at FAB.WHO 3.624650711 0.004850853 0.021482349EPOR 209962_at FAB.WHO 7.98483222 8.80E-06 5.46E-05EPOR 209963_s_at FAB.WHO 7.0868934 2.77E-05 0.000156067EPOR 215054_at FAB.WHO 4.217561471 0.001825108 0.009429725EPOR 216999_at FAB.WHO 3.40148799 0.007078019 0.027427324EPOR 37986_at FAB.WHO 10.03434177 8.17E-07 6.33E-06EPO 217254_s_at FAB.WHO 3.900318986 0.003064263 0.014614176


GeneSymbol probeset_ID param Fval p-val p-val.adjEPO 207257_at FLT3_ITD 5.413039736 0.011843388 0.054141204CSF3 207442_at FLT3_ITD 5.238869247 0.013340583 0.056919822EPOR 209963_s_at FLT3_ITD 4.422716217 0.023704341 0.084282101TPO 210342_s_at FLT3_ITD 6.022128682 0.007884537 0.042050866MPL 216825_s_at FLT3_ITD 8.594821799 0.001631388 0.010440885EPOR 216999_at FLT3_ITD 5.839249444 0.008895631 0.043793878


GeneSymbol probeset_ID param Fval p-val p-val.adjEPOR 209962_at aml 2.470916665 0.057784563 0.176105335TPO 210342_s_at aml 2.412645021 0.062616381 0.182156746MPL 211903_s_at aml 3.198583323 0.022029413 0.082934262EPOR 37986_at aml 6.909845027 0.000448428 0.002207647EPOR 396_f_at aml 2.864195149 0.03400293 0.120899305


GeneSymbol probeset_ID dep Fval p-val p-val.adjCSF3R 1553297_a_at karyotype 14.64739264 9.77E-16 6.66E-15CSF3R 203591_s_at karyotype 14.57918633 1.14E-15 6.68E-15MPL 207550_at karyotype 13.68382674 9.16E-15 4.91E-14EPOR 209962_at karyotype 3.885942457 0.000507697 0.001010326TPO 210342_s_at karyotype 2.144506009 0.040079994 0.053678564MPL 211903_s_at karyotype 3.937070914 0.000444442 0.000925921EPOR 215054_at karyotype 3.314382018 0.002221146 0.003874092MPL 216825_s_at karyotype 3.497965943 0.001386359 0.002536022EPOR 37986_at karyotype 2.742171075 0.00942085 0.014720077EPOR 396_f_at karyotype 7.63683026 2.77E-08 7.69E-08


Data set 4 GSE13159http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13159Gene probeset_ID param Fval p-val p-val.adjCSF3R 203591_s_at leukemia_class 6.091798729 0.014336512 0.034305226KIT 205051_s_at leukemia_class 35.99080484 7.98E-09 5.34E-08EPOR 396_f_at leukemia_class 38.93745324 2.19E-09 1.63E-08MPL 207550_at leukemia_class 5.402500921 0.021011631 0.048544113EPOR 215054_at leukemia_class 67.7921504 1.55E-14 2.08E-13EPOR 37986_at leukemia_class 57.46026308 9.29E-13 8.90E-12EPOR 209963_s_at leukemia_class 66.41570917 2.65E-14 2.96E-13EPOR 209962_at leukemia_class 74.66373288 1.11E-15 1.86E-14CSF3 207442_at leukemia_class 3.418986398 0.065780102 0.12592191MPL 216825_s_at leukemia_class 4.157811832 0.042627775 0.089251904


Supplementary Table 5. Frequency of Pro-Glu-Pro-Glu insertion in HCLS1

protein in AML patients

Pro-Glu-Pro-Glu insertion AML Patients Controls OR P*

Genotype frequency 0.0001389

Pro-Glu-Pro-Glu -/- 46 (34,1 %) 63 (60,5 %) 0,56

Pro-Glu-Pro-Glu +/- 83 (61,5 %) 39 (37,5 %) 1,64

Pro-Glu-Pro-Glu +/+ 6 (4,4 %) 2 (1,9 %) 2,31

Allele frequency 0.0005313

Pro-Glu-Pro-Glu - 175 (64,8 %) 165 (79 %) 0,82

Pro-Glu-Pro-Glu + 95 (35,2 %) 43 (21,5 %) 1,7

OR = odds ratio.

* Distribution in patients versus controls, by chi-square test.


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