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SUPPLEMENTARY MATERIALS

SUPPLEMENTARY EXPERIMENTAL PROCEDURES

Long-term reconstitution assay: Two to four month old adult recipient mice were irradiated

with a Gammacell 40 Exactor Cesium137 γ-ray source (MDS Nordia, ON, Canada) delivering

approximately 110 rad/min. Lethal irradiation was done by giving two doses of 540 rad,

delivered at least 2 hours apart, and sublethal irradiation was performed by giving a single dose

of 600 rad. For transplantation of virus-infected bone marrow or hematopoietic stem/progenitor

cells into lethally irradiated primary recipients, donor (CD45.1) cells and 200,000 recipient

(CD45.2) radioprotective bone marrow cells were resuspended in 100 µl of staining medium and

injected into the retro-orbital venous sinus of individual lethally irradiated recipient mice.

To test the reconstituting potential of Sox17-expressing hematopoietic progenitors

isolated from primary recipients, 50 GFP+ donor CD48+LSK cells were double sorted into

individual wells of a 96-well plate containing 200,000 recipient whole bone marrow cells in

staining medium. The contents of individual wells were injected into the retro-orbital venous

sinus of individual lethally irradiated secondary recipients. To test the reconstituting potential of

wild type adult bone marrow CD48+LSK cells, 100, 500 or 2,500 double sorted CD48+LSK cells

from 8-week old UBC-GFP mice (CD45.2) were transplanted into sublethally irradiated (600

rads) CD45.1 recipients. The use of sublethally irradiated recipients allowed monitoring of donor

cell engraftment as early as one week after transplantation. To monitor donor cell engraftment,

blood was obtained from the tail veins of recipient mice every 1 to 4 weeks for at least 16 weeks

after transplantation, blood cells were subjected to ammonium-chloride potassium red cell lysis

(Morrison and Weissman 1994), and stained with directly conjugated antibodies against

CD45.2-Alexa Fluor 700 (104), CD45.1-APC/Efluor 780 (A20), B220-PE/Cy5 (RA3-6B2), Mac-1-

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PE (M1/70), CD3e-APC (145-2c11), and Gr-1-PE/Cy7 (RB6-8C5) to examine donor cell

chimerism.

Generation of MSCV-Sox17 and MSCV-Sox17his retrovirus: To generate C-terminal 6xhis

tagged mouse Sox17, full length mouse Sox17 cDNA was PCR amplified from mouse brain

cDNA. AseI and XhoI sites were added to the 5’ and 3’ ends respectively and the sequence was

inserted into NdeI/XhoI digested pET23b vector (EMD chemicals, Gibbstown, NJ) to generate

the pET23-mSox17-6xhis vector. A Sox17-6xhis fragment was then isolated using FspI/BsaAI

double digestion and inserted into HpaI digested mouse stem cell virus (MSCV) based pMIG

(MSCV-GFP) vector (Van Parijs et al. 1999), immediately upstream of the internal ribosome

entry site (IRES) to generate the MSCV-Sox17his vector. The MSCV-Sox17 vector was

generated similarly except a short linker containing an in-frame stop codon and a FspI site was

inserted into the pET-23-mSox17-6xhis vector before the 6xhis tag to generate pET23-

mSox17STOP, and a FspI fragment containing Sox17 was then inserted into HpaI digested

pMIG to generate the MSCV-Sox17 vector.

Preparation of high-titer virus was carried out by transfecting 293T cells with control

(MSCV-GFP) or Sox17-encoding MSCV vector together with the pCL-Eco packaging vector

(Naviaux et al. 1996) using standard calcium phosphate methods as previously described (He et

al. 2009). Cells were allowed to produce virus for 24-48 hours, then viral supernatants were

collected, 0.45 micron filtered, and stored at –80°C. Virus titer was determined by infecting

2x105 3T3 cells using 1, 10 or 100 µl of viral supernatant. A viral titer of 2-5x106/ml was routinely

obtained.

Cytology, histology and immunohistochemistry: For Wright-Giemsa staining of blood

smears, bone marrow, spleen, and liver cells, freshly prepared blood smears and cytospun

slides were air-dried, fixed in methanol for 5 min, stained in Wright-Giemsa staining solution

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(Sigma, St. Louis, MO) for 2 minutes, developed in 8.3mM phosphate buffer (pH=6.8) for 2-5

min, dipped 10 times in deionized water, dehydrated through an ethanol gradient and mounted

in Permount (Fisher Scientific, Waltham MA).

For histological analysis of hematopoietic tissues, spleen, liver, lung and lymph node

samples were fixed in 10% neutral buffered formalin and paraffin embedded. Five micron

sections were cut on a Leica microtome and stained with hematoxylin and eosin following

standard protocols. Immunohistochemical staining for Von Willebrand’s Factor (VWF) on tissue

sections was performed using a DAKO Autostainer (DAKO, Carpinteria, CA) using DAKO

Envision+ and diaminobenzadine (DAB) as the chromogen. De-paraffinized sections of formalin

fixed tissue at 5µm thickness were labeled with rabbit polyclonal anti-VWF (1:500, DAKO) for 60

minutes after microwave citric acid epitope retrieval. Immunofluorescent staining of VWF on

sorted GFP+CD41+ cells was performed by fixing the sorted cells in 4% paraformaldehyde in 1x

phosphate buffered saline (PBS) for 10 min on ice, followed by washing three times with 1x PBS

and cytospinning onto microscope slides. Rehydrated slides were blocked/permeabilized with

1xPBS containing 1% Triton X-100 and 10% goat serum. Slides were stained with anti-VWF

(1:500) at 4°C overnight. After washing, slides were incubated with secondary antibodies

conjugated with Alexa Fluor 555 (Invitrogen) together with DAPI for 1 hour at room temperature.

All images were collected using an Olympus BX-71 inverted microscope with 20x and 40x dry

objectives. Histology was analyzed by a hematopathologist and classified according to the

Bethesda protocols for the classification of hematopoietic neoplasms in mice (Kogan et al. 2002;

Morse et al. 2002).

Microarray analysis: Total RNA (~5ng) was isolated from 3-5 independent, freshly isolated

aliquots of 10,000 E16.5 fetal liver HSCs, 10,000 fetal liver CD48+LSK cells, 10,000 adult bone

marrow HSCs, 10,000 adult bone marrow CD48+LSK cells, 10,000 Sox17-expressing

CD48+LSK cells isolated from primary recipients 12 weeks after transplantation of MSCV-

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Sox17-infected bone marrow cells, or 10,000 HSCs isolated from 8-week old Sox17-transgenic

mice that were treated with doxycycline for 5 days to induce transgene expression. Total RNA

was extracted using Trizol with 25 µg/ml linear acrylamide (Ambion, Austin, TX). The extracted

RNA (10 µl volume) was treated with 2 Units of RNase-free recombinant DNase I (Ambion) for

15 min at 25˚C, followed by 15 min at 37˚C to remove any contaminating genomic DNA. The

DNA-free RNA samples were purified using the RNA Clean & Concentrator™-5 from Zymo

Research (Orange, CA) and eluted in 10 µl of nuclease-free water. Purified RNA was reverse

transcribed and amplified using the WT-Ovation™ Pico RNA Amplification system (NuGEN

Technologies, San Carlos, CA) following the manufacturer’s instructions. Sense strand cDNA

was generated using WT-Ovation™ Exon Module (NuGEN), and fragmented and labeled using

the FL-Ovation™DNA Biotin Module V2 (NuGEN). 2.5 µg of labeled cDNA were hybridized to

Affymetrix Mouse Gene ST 1.0 microarrays. The chips were hybridized and scanned according

to the manufacturer's instructions. Expression values for all genes were calculated using the

robust multi-array average (RMA) method (Irizarry et al. 2003).

For the identification of genes with differential expression between groups, the raw

expression data were analyzed using Expander v5.1 software (Shamir et al. 2005), and genes

with fold changes greater than 2 and p-values less than 0.05 (t-test, using log2 transformed

expression values) between sample groups were considered to be significantly changed. The

complete list of differentially expressed genes between Sox17-expressing and control

CD48+LSK cells is in Suppl. Table 3; the complete list of differentially expressed genes between

Sox17-transgenic and control HSCs is in Suppl. Table 4

For Gene Set Enrichment Analysis (GSEA), normalized expression data were assessed

by GSEA 2.0.6 software as described (Mootha et al. 2003; Subramanian et al. 2005). The

complete gene expression profiles of Sox17-expressing and control CD48+LSK cells (or Sox17-

transgenic and control HSCs) were used as the background data set and the following gene

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sets were used as the foreground: complete C2-curated gene sets from the Broad Institute

(http://www.broadinstitute.org/gsea/index.jsp), WNT-signaling gene set and WNT-targets gene

set curated by the Broad Institute, HSC or short-term progenitor (CD48+LSK cell)-specific gene

sets, and a fetal liver HSC-specific gene set. We generated HSC or short-term progenitor

(CD48+LSK cell)-specific gene sets by comparing adult bone marrow HSCs versus bone

marrow CD48+LSK cells (p<0.05, fold change >2). We generated a fetal liver HSC-specific gene

set by comparing fetal liver HSCs versus adult bone marrow HSCs (p<0.05, fold change >2).

See Suppl. Table 2 for the complete list of genes included in the cell type-specific gene sets.

Gene Ontology (GO) and Pathway analysis were performed in Expander v5.1 software

(Shamir et al. 2005). Enrichement of GO terms or KEGG pathways were searched in three sets

of genes: genes that were significantly changed for more than 2-fold in CD48+LSK progenitors

after viral Sox17 over-expression, genes that were significantly changed in HSCs after

transgenic Sox17 over-expression, and genes that are significantly changed for more than 2-

fold in both circumstances. The lists of significantly enriched GO terms and KEGG pathways

downstream of Sox17 were summarized in Suppl. Table 5.

Quantitative RT-PCR: Quantitative (real-time) RT-PCR was performed as described previously

(Molofsky et al. 2003). Three replicates of 10,000 E16.5 fetal liver HSCs, 10,000 HSCs from

Sox17-transgenic mice that were administered with doxycycline for 5 days, and 10,000 Sox17-

expressing or control CD48+LSK cells from reconstituted primary recipients were sorted into

Trizol. RNA was isolated using chloroform extraction and isopropanol precipitation. cDNA was

made with random primers and SuperScript III reverse transcriptase (Invitrogen). Quantitative

PCR was performed with cDNA from 100-500 cell equivalents using a SYBR Green Kit and a

LightCycler 480 (Roche Applied Science). Primers used to amplify Sox17 and microarray

candidate genes are listed in Suppl. Table 1.

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Western blotting: Western blotting was performed as previously described (He et al. 2009).

For western blotting of protein from virus infected 3T3 cells and whole splenocytes, MSCV-

GFP, MSCV-Sox17 or MSCV-Sox17his virus infected 3T3 cells, or whole splenocytes isolated

from primary recipients reconstituted with control or Sox17 virus infected whole bone marrow

cells were homogenized in ice-cold RIPA buffer supplemented with protease inhibitor cocktail

(complete mini tablet, Roche), by brief sonication using a Virsonic-100 ultrasonic cell disruptor

(VirTis; Gardiner, NY). The cell homogenates were then spun down for 10 minutes at 4°C at

16,000g and the supernatant was quantified colorimetrically (BioRad protein assay; Bio-Rad,

Hercules, CA). 10 µg of protein per lane were separated on a 4-20% gradient Bis-Tris gel and

transferred for 2 hours to PVDF membranes (Bio-Rad) on ice. For western blotting of protein

from flow cytometrically isolated LSK cells, 50,000 E12 LSK cells, 50,000 E13.5 LSK cells,

50,000 LSK cells from primary recipients of MSCV-Sox17 or MSCV-Sox17his virus infected

adult hematopoietic cells, 50,000 LSK cells from Sox17-transgenic mice that were administered

doxycycline for 5 days, and 50,000 LSK cells from control adult mouse bone marrow were

double sorted into ice-cold staining medium. Cells were pelleted by centrifugation at 2000xg for

10 min, washed once with 1xPBS to remove residual serum proteins, then homogenized in ice-

cold RIPA buffer supplemented with protease inhibitor cocktail. The total protein was separated

on a 4-20% gradient Bis-Tris gel and transferred for 2 hours to PVDF membranes (Bio-Rad) on

ice. Primary antibodies were mouse monoclonal anti-β-actin (A1978; Sigma), polyclonal goat-

anti-SOX17 (AF1924; R&D system, Minneapolis, MN), and rabbit polyclonal anti-histone H3

(ab1791; Abcam, Cambridge, MA). Horseradish peroxidase (HRP)-conjugated secondary

antibodies, and SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific,

Rockford, IL) were used to visualize the primary antibody staining. Quantification of SOX17

protein expression level in each sample was performed by scanning the western blots, and

measuring the density of respective protein bands in ImageJ software (http://rsbweb.nih.gov/ij/).

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Statistics: All experiments were performed at least three times and all data are expressed as

mean±SD. Statistical comparisons between samples were performed in Excel using Student’s t-

test. Unless otherwise indicated, a p-value less than 0.05 was considered statistically

significant. The survival analysis of primary and secondary recipients was performed in

Graphpad Prism 5 software, and the p-values for log-rank test between individual curves are

presented.

SUPPLEMENTARY FIGURE LEGENDS

Supplementary figure 1: Infection by MSCV-Sox17 or MSCV-Sox17his retrovirus, but not

MSCV-GFP control retrovirus, leads to increased Sox17 expression. A). Schematic

representations of MSCV-GFP, MSCV-Sox17 and MSCV-Sox17his viruses. B) Western blotting

for SOX17 protein in control or Sox17 virus infected 3T3 cells, or splenocytes isolated from

primary recipients reconstituted with control or Sox17 virus infected bone marrow cells. SOX17

protein could only be detected in cells infected with MSCV-Sox17 or MSCV-Sox17his virus, but

not in cells infected with MSCV-GFP virus. Note that multiple bands were observed on the

SOX17 western blots and that the number and precise migration of the bands varied somewhat

among different cell types, suggesting that SOX17 may undergo different post-translational

modifications or may use alternative start sites in different cell types (Kanai et al. 1996). C).

Quantitative RT-PCR comparing the expression level of Sox17 transcripts in E16.5 fetal liver

HSCs, LSK cells isolated from primary recipients of MSCV-Sox17 or MSCV-Sox17his virus

infected adult hematopoietic cells, and HSCs isolated from Sox17-transgenic mice treated with

doxycycline for 5 days. D) Western blotting for SOX17 protein in 50,000 flow cytometrically

isolated E12 fetal liver LSK cells, E13.5 fetal liver LSK cells, LSK cells isolated from primary

recipients of MSCV-Sox17 or MSCV-Sox17his virus infected adult hematopoietic cells, LSK

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cells isolated from adult Sox17 transgenic mice treated with doxycycline for 5 days, and LSK

cells isolated from control adult mice. Multiple protein bands with sizes between 50 and 60 kD

(as expected for SOX17 (Kanai et al. 1996)) were detected in E12 and E13.5 LSK cells and in

adult LSK cells after ectopic Sox17 over-expression but not in control adult LSK cells. It was

unclear whether the different sizes of the SOX17 protein bands detected in different samples

reflected different post-translational modifications of SOX17 protein in fetal and adult

hematopoietic cells. Numbers on top of each lane quantitate band intensity in each lane as

measured by densitometry relative to E12 fetal liver LSK cells. Numbers without parentheses

indicate quantification based on only the bottom two bands. Numbers within parentheses

indicate quantification of all bands.

Supplementary figure 2: Representative flow-cytometry plots demonstrating the gating

schemes used to detect donor (GFP+) red blood cells (RBCs), platelets (PLT), myeloid

cells (Mac-1+ or Gr-1+), B cells (B220+) or T cells (CD3+) in the peripheral blood of primary

recipient mice at 16 weeks after transplantation. A-G) Representative flow-cytometry plots

showing GFP+ blood cells in primary recipient mice reconstituted by uninfected, MSCV-GFP-

infected, or MSCV-Sox17-infected bone marrow cells. Note the lower expression of Gr-1 by

Sox17-expressing Mac-1+ myeloid cells, similar to myeloid lineage cells in fetal liver (Fig. 3F).

For erythroid cell (RBC) and platelet (PLT) analysis, whole blood (unlysed) was used. For

analysis of all white blood cell (WBC) lineages, red blood cells were lysed prior to antibody

staining.

Supplementary figure 3: Adult bone marrow CD48+LSK cells were only able to give

transient multilineage reconstitution of irradiated mice. A-F) 100, 500 or 2500 adult bone

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marrow CD48+LSK cells from UBC-GFP mice were transplanted into 600 rad sublethally

irradiated recipients, and peripheral blood GFP+ cell chimerism was monitored up to 16 weeks.

Adult CD48+LSK cells gave transient multilineage reconstitution of the myeloid (A, B; Mac-1+ or

Gr-1+), B (C; B220+), T (D, CD3+), erythroid (E, Ter119+) and platelet (F, CD41+) lineages. G)

Summary of recipient reconstitution patterns by donor cells.

Supplementary figure 4: Sorting strategy for isolation of adult hematopoietic stem and

progenitor cell populations as well as viral infection efficiency. A-E) Sorting strategy for

isolation of adult bone marrow HSCs, MPPs, CD48+LSK cells, GMPs, and Pre-MegE cells. F)

Viral infection efficiency of cultured adult hematopoietic stem and progenitor cells as determined

at 48 hours after infection.

Supplementary figure 5: Ectopic Sox17-expression significantly increased the long-term

reconstituting potential of adult HSCs but not GMPs or Pre-MegE progenitors. A) 500

control or MSCV-Sox17-infected adult bone marrow HSCs were transplanted into lethally

irradiated recipients along with 200,000 recipient cells. Sox17-expressing HSCs showed

significantly increased long-term reconstitution in the myeloid (Mac-1+ or Gr-1+), erythroid

(Ter119+), and platelet (CD41+) lineages as compared to control virus infected cells at 16 weeks

after transplantation (*, p<0.05). The number to the right of each plot indicates the percentage of

cells within the indicated lineage that were GFP+ (mean±SD) at 16-weeks after transplantation.

Black lines represent individual mice infected with control virus while green lines indicate

individual mice infected with MSCV-Sox17 virus. B, C) 500 control or MSCV-Sox17-infected

bone marrow GMPs or Pre-MegEs were transplanted into lethally irradiated recipients along

with 200,000 recipient cells. Neither control nor Sox17-expressing cells from these populations

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gave detectable donor cell reconstitution in peripheral blood between 4 and 16 weeks after

transplantation. The numbers of recipients in each experiment can be found in Fig. 6C.

Supplementary figure 6: Sox17-expressing HSCs, MPPs, and CD48+LSK cells were

capable of giving long-term multilineage reconstitution in secondary recipients. A)

200,000 whole bone marrow cells from primary recipients of MSCV-Sox17 virus-infected HSCs,

MPPs, or CD48+LSK cells, together with 200,000 recipient cells were transplanted into

irradiated secondary recipient mice. The contribution of GFP+ donor cells to peripheral blood

was assessed 16 weeks after transplantation. B). Three of three secondary recipients of whole

bone marrow cells isolated from primary recipients of MSCV-Sox17 virus-infected HSCs

exhibited long-term reconstitution in the myeloid, erythroid, and platelet lineages. Five of 10

secondary recipients of whole bone marrow cells from primary recipients of MSCV-Sox17 virus-

infected MPPs exhibited long-term multilineage reconstitution in all blood lineages. Four of 4

secondary recipients of whole bone marrow cells from primary recipients of MSCV-Sox17 virus-

infected CD48+LSK cells exhibited long-term multilineage reconstitution in all blood lineages.

Supplementary figure 7: Ectopic Sox17-expression in adult CD48+LSK cells increased the

expression of genes preferentially expressed by HSCs and reduced the expression of

genes preferentially expressed by restricted progenitors in gene sets independently

generated by others. A-D) GSEA profiles comparing Sox17-expressing (red) and control bone

marrow (blue) CD48+LSK cells using C2-curated gene sets from the Broad Institute

(http://www.broadinstitute.org/gsea/index.jsp). Sox17-expressing CD48+LSK cells showed

significant enrichment of genes preferentially expressed by long-term self-renewing HSCs (as

compared to whole bone marrow cells (A) and depletion of genes that are preferentially

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expressed by various fractions of hematopoietic progenitors at various stages of maturation (as

compared to whole bone marrow cells (B-D). These gene sets were assembled by the Broad

Institute based on microarray data published by Ivanova et al. (Ivanova et al. 2002). E-F) GSEA

profiles comparing Sox17-expressing (red) and control bone marrow (blue) CD48+LSK cells

using gene sets for WNT pathway components and WNT target genes curated by the Broad

Institute. Neither gene set was significantly enriched in either the Sox17-expressing or control

CD48+LSK cells.

Supplementary figure 8: Ectopic Sox17 expression in Sox17-transgenic HSCs increased

the expression of genes associated with fetal and adult HSCs and reduced the

expression of genes associated with restricted progenitors. A). Short-term Sox17-

transgene expression significantly increased the frequency of LSK cells in the bone marrow and

spleen of Sox17-transgenic mice. B-D) GSEA profiles comparing HSCs isolated from Sox17-

transgenic (red) and control (blue) mice after 5 days of doxycycline administration. Sox17-

transgenic HSCs showed significant enrichment of fetal HSC (compared to adult bone marrow

HSCs) and adult HSC (compared to bone marrow CD48+LSK cells) gene sets (B, C), and

significant depletion of a progenitor (CD48+LSK cell compared to HSCs) gene set (D). NES

stands for normalized enrichment score. E) Comparison of the gene expression profiles of

Sox17-transgenic and control HSCs revealed 97 significantly upregulated and 7 significantly

down-regulated genes in the Sox17-expressing cells (fold change >2 and p<0.05). Eleven of the

97 upregulated genes were preferentially expressed by fetal HSCs (compared to adult HSCs,

fold >2, p<0.05), while 6 were preferentially expressed by adult HSCs (compared to fetal HSCs,

fold >2, p<0.05); four of the 7 downregulated genes were also downregulated in fetal HSCs

(compared to adult HSCs, fold >2, p<0.05). The complete list of significantly up and down

regulated genes in Sox17-transgenic HSCs can be found in Suppl. Table 4.

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Supplementary Figure 9: Long-term ectopic Sox17 expression in adult hematopoietic

cells induced nonlymphoid leukemias. A). H&E staining of representative tissue sections

from primary recipient mice in each treatment. Infiltration of blast cells could be seen in spleen,

liver and lung, resulting in the disruption of normal tissue architecture. Increased numbers of

erythroid cells at various stages of differentiation could also be seen in the spleens of Sox17-

expressing cell recipients (inset). B). The infiltrating blast cells in the spleens of recipients

transplanted with Sox17-expressing cells stained positively for Von Willebrand factor (VWF+,

brown), suggesting they arise from the megakaryocyte lineage. Only resident megakaryoctes

and platelets stained positively for VWF in control spleen sections. C) Wright-Giemsa staining of

cytospin preparations of bone marrow, spleen, and/or liver cell samples from moribund

recipients of Sox17-expressing or control cells showed the presence of leukemic blast cells and

expanded erythroid cells. D) Sox17-expressing GFP+c-kit+CD41+ cells had a morphology similar

to the leukemia blast cells and were also VWF+.

SUPPLEMENTARY TABLES

Supplementary table 1: List of qRT-PCR primers used to assess the expression of

candidate genes from the microarray.

Supplementary table 2: List of customized gene sets used for gene set enrichment

analysis. All gene sets were generated by comparing the global gene expression profiles

between each pair of indicated cell populations, and selected for genes that are significantly

changed (p<0.05, t-test) by more than 2-fold between samples.

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Supplementary table 3: Significantly upregulated and downregulated genes in Sox17-

expressing CD48+LSK cells as compared to control bone marrow CD48+LSK cells. Only

genes that were significantly (p<0.05) changed by more than 2-fold between sample groups

were listed. Whether each gene was significantly up or down regulated in HSCs (versus bone

marrow CD48+LSK cells) or fetal HSCs (versus adult bone marrow HSCs) is also indicated.

Supplementary table 4: Significantly upregulated and downregulated genes in Sox17-

transgenic HSCs as compared to littermate control HSCs after 5 days of doxycycline

administration. Only genes that were significantly (p<0.05) changed by more than 2-fold

between sample groups were listed. Whether each gene was significantly up or down regulated

in HSCs (versus bone marrow CD48+LSK cells) or fetal HSCs (versus adult bone marrow

HSCs) is indicated. Whether each gene was significantly up or down regulated in CD48+LSK

cells after retroviral over-expression of Sox17 is also indicated.

Supplementary table 5: Significantly enriched Gene Ontology (GO) terms and KEGG

pathways downstream of Sox17. Only GO terms and KEGG pathways that were significantly

(p<0.05, and p<0.01 respectively) enriched in genes that were changed in CD48+LSK cells

and/or HSCs after ectopic Sox17 expression are listed. Eight KEGG pathway maps were

enriched in genes that were significantly changed in CD48+LSK progenitors after viral Sox17

over-expression, and 2 KEGG pathway maps were enriched in genes that were significantly

changed in HSCs after transgenic Sox17 over-expression. Notably, genes from the “Cytokine-

cytokine receptor interaction” KEGG pathway were significantly enriched in both comparisons.

We identified 65 GO terms that were enriched in genes that were significantly changed in

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CD48+LSK progenitors after viral Sox17 over-expression, 13 GO terms that were enriched in

genes that were significantly changed in HSCs after transgenic Sox17 over-expression, and 9

GO terms that were enriched in genes that were significantly changed in both circumstances.

“Regulation of signal transduction” and “regulation of cell proliferation” were enriched GO terms

in both circumstances after ectopic Sox17 expression.

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