SUPPLEMENTAL MATERIAL AND METHODS
Cell Lines and culture:
Cell lines were maintained at 37° C in a 5% CO2 atmosphere. The A673, TC32, and
EW8 cell lines were kindly provided by Dr. Kimberly Stegmaier (Dana-Farber Cancer
Institute, Boston, MA). The BJ-tert, HEK-293T and U2OS cell lines were obtained from
ATCC. A673, EW8, BJ-tert, HEK-293T and U2OS cells were grown in Dulbecco’s
Modified Eagle’s Media (DMEM) supplemented with 10% FBS, 100 IU ml−1 penicillin
and 100 μg ml−1 streptomycin. TC32 cells were grown in RPMI supplemented with 10%
FBS, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin.
The EF+ and EFFib cells were cultured on gelatin-coated plates. EF+ cells can be grown on
uncoated plates, but the initial adherence after passaging is decreased relative to the EFFib
cells. For passaging, the cells were treated with versene (Gibco) for 5 min at 37° C and
then gently collected using a cell scraper (Corning). Cells were pelleted at 300 r.c.f. and
then re-suspended in DMEM supplemented with 10% FBS, 1 gml-1 doxycycline,
100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. EF+ cells were used in experiments
after passage 20.
Design and construction of plasmids:
EWS-FLI1 was PCR amplified using Q5 High-Fidelity Polymerase (New England
Biolabs) from a pcDNA-EWS-FLI1 plasmid (gift from Dr. A. Kung) and transferred into
the MCS-1 site of the tetracycline-inducible pLVX-TRE3G-IRES vector (Clontech)
using In-Fusion (Clontech) cloning. GFP was then cloned into the MCS-2 site,
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downstream of the IRES, using In-Fusion cloning. Finally, the U6 promoter and a TP53
shRNA was PCR amplified from the plasmid TRCN0000003755 (Sigma-Aldrich) and
cloned into the XhoI site of the plasmid using In-Fusion.
Lentivirus production and infection:
Lentivirus was produced by transfecting HEK-293T cells with the pLVX-EF1-IRES-GFP
vector or the pLVX-EF1a-Tet3G vector and packaging plasmids (psPAX2 and pMD2.G)
according to the FuGENE 6 (Roche) protocol. Virus was concentrated 50-fold with Peg-it
virus precipitation solution (System Biosciences). For lentiviral transduction, H1 stem
cells were incubated with 10-40 l of concentrated virus in mTESR1 media and 5 g/mL
of polybrene (Sigma-Aldrich) for 12-16 hours. Cells were sequentially infected and
selected in 1 g/mL puromycin (pLVX-EF1-IRES-GFP) or 500 g/mL geneticin (pLVXEF1a-Tet3G) 48 hours after transduction.
Flow Cytometry:
Cells were incubated with antibodies for one hour at 4 °C and then washed twice with
FACS buffer (2% FBS in PBS). After the last wash, the cells were re-suspended in 300500 l of FACS buffer and incubated on ice until used in the flow cytometry experiment.
Flow cytometry was performed on a FACSCalibur Instrument (BD Biosciences) and data
were analyzed using CellQuest Pro (BD Biosciences). A minimum of 50,000 events was
collected. The following antibodies were used for flow cytometry: PE Mouse IgG1
Isotype Reagent (MOPC-21; BD Biosciences, 55574), APC Mouse IgG1 Isotype
Reagent (MOPC-21; BD Biosciences 555751), APC Mouse IgG2a Isotype Reagent
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(X39; BD Biosciences, 340757), CD34-APC (581; BD Biosciences, 555824), CD73-PE
(AD2; BD Biosciences, 550257), CD99-APC (HI156; Abcam, ab91292), CD105-APC
(266; BD Biosciences, 562408), CD117-APC (104D2; eBioscience, 8017-1178) and
CD271-PE (C40-1457; BD Biosciences, 560927).
Fluorescent in-situ hybridization:
Cells were collected with versene, washed with PBS and then re-suspended in 0.56% KCl
for 20 min followed by fixation in 3:1 methanol acetic acid. After six methanol:acetic
acid washes, the cells were transferred to coverslips and incubated in 2x SSC plus 0.5%
NP-40 for 30 min at 37 °C. The cells were then dehydrated in 70%, 85% and 100%
ethanol washes. FISH probes for chromosomes 6 and 8 (Cytocell) were diluted 1:10 in
hybridization buffer (Cytocell) and added to the coverslips. The coverslips were affixed
to slides with rubber cement and then placed in a hybridization oven at 75 °C for 5 min,
followed by an overnight incubation at 37 °C. The cells were then briefly washed in PBS
to remove the rubber cement followed by incubation in wash buffer (0.5× SSC plus 0.1%
SDS) for 5 min at 65 °C. Coverslips were then incubated with PBS plus 0.2 µg ml-1 DAPI
before being sealed on a slide with mounting medium.
Immunoblots: Whole-cell extracts for immunoblotting were prepared by incubating cells
in RIPA buffer (Boston BioProducts) plus protease inhibitors (Complete, Mini, EDTAfree. Roche) for 20 min. Supernatants were collected following a 15 min centrifugation at
17,000 r.c.f. at 4 °C. Protein concentrations were determined using the BCA reagent
(Pierce). SDS-PAGE was used to separate proteins, which were then transferred to
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polyvinylidene difluoride membranes (Millipore). Antibodies to the following proteins
were used in the immunoblots: p53 (DO-1, Santa Cruz, sc-126, 1:500), FLI1 (C-19, Santa
Cruz, sc-356, 1:1000) and Vinculin (Cell Signaling, #4650, 1:1000).
Viral Integration Site Analysis:
Lentiviral integration site analysis was performed using the Lenti-X Integration Site
Analysis Kit (Clontech), according to the manufacturer’s instructions.
Cell Proliferation Assay:
Cells were plated in triplicate in 6-well plates and collected with trypsin at different time
points. Live cells were then counted with a Vi-CELL Cell Viability Analyzer (Beckman
Coulter).
Cell proliferation and viability were also measured using CellTiter-Glo (Promega).
Luminescence readings were obtained using a FLUOstar Omega microplate reader (BMG
Labtech). For experiments with drug treatment, approximately 5 x 106 cells were plated
per well of a 96-well plate. Cells were treated with a range of drug concentrations for
three days. IC50 values were then calculated using log-transformed and normalized data
(GraphPad Prism 5.0).
Quantitative reverse transcriptase PCR: Total RNA was extracted using RNAeaasy kit
following the manufacturer's instructions. 1 μg of total RNA was reverse-transcribed into
first-strand cDNA using random hexamer primers and the SuperScript III Reverse
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Transcriptase (Invitrogen). RT-qPCR was performed on the ViiA 7 Real-Time PCR
System (Life Technologies) using SYBR Select Master Mix (Life Technologies).
Reactions were performed in triplicate and gene expression was normalized to actin. PCR
primer sequences are listed in Supplemental Table S1.
Soft Agar Assay:
Cells (2×104) were plated in triplicate in 6-well plates with 0.6% base agar and 0.3% top
agar and incubated for fourteen days.
Contact inhibition assay:
Cells were allowed to grow for fourteen days in 10 cm dishes and then fixed with 4%
paraformaldehyde in PBS for 15 min. Colonies were stained for ten minutes with 0.5%
methylene blue and 1% ethanol in PBS. After staining, the plates were washed four times
with PBS.
Short Tandem Repeat (STR) Profiling:
STR profiling of cell lines was performed using the GenePrint 10 System (Promega) to
ensure that the EF+ cells were derived from the parental, H1 stem cell line and not due to
contamination by Ewing sarcoma cell lines, or other transformed cell lines (Supplemental
Table S2).
Xenograft:
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Approximately 3.0 x 106 or 1 x 107 cells were mixed with 30% matrigel and injected
subcutaneously into the flanks of female nod scid gamma (NSG) mice. Eight mice were
injected in each group. Five mice were injected with 3.0 x 106 TC32 cells as a positive
control for tumor growth. Mice were fed normal chow or chow containing doxycycline
(2000 part per million). Tumor growth was assessed using palpation and caliper
measurements.
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SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure S1. Expression of EWS-FLI1 in WA25 stem cells generates
properties of transformation. (A) Western blot of EWS-FLI1 in the WA25-EF+ cells and
the WA25-EF- cells after four days of doxycycline removal. (B) The immunophenotype
of WA25-EF+ cells as determined by flow cytometry. Cells were labeled with APC- and
PE-labeled antibodies against CD34, CD73, CD99, CD105, c-Kit, CD271 and isotypematched control antibodies. The isoptype control is shown in black and the specific
antibody is shown in red. (C) Relative growth of WA25-EF+ cells and WA25-EF- cells
after seven days without doxycycline. Error bars indicate the standard error of the mean
of three experiments (*, p<0.05). (D) The WA25-EFFib and WA25-EF+ cells were
cultured for fourteen days without passaging and then colonies were stained with
methylene blue. (E) Soft-agar assay for anchorage-independent growth of WA25-EFFib
and WA25-EF+ cells. Error bars indicate the standard error of the mean of three
experiments (*, p<0.05).
Supplemental Figure S2. (A) The morphology of the embryoid bodies demonstrates a
uniform size and shape. (B) RT-qPCR shows the expression of lineage markers from the
three germ layers in the embryoid bodies. Data show the fold change in gene expression
between the embryoid bodies and stem cells. Error bars indicate the standard error of the
mean of three experiments. (C) Dissociation of the embryoid bodies with trypsin to single
cells followed by analysis with flow cytometry for GFP, a marker of EWS-FLI1
expression, and 7-AAD, a marker of dead cells.
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Supplemental Figure S3. Growth of EFFib cells compared to EFFib cells after the addition
of doxycycline to the cell culture medium. Cell growth was measured seven days after
adding doxycycline using trypan blue exclusion and a Vi-CELL Cell Viability Analyzer.
Error bars indicate the standard error of the mean of three experiments (*, p<0.05).
Supplemental Figure S4. Karyotype analysis. (A) G-band karyotype analysis of EFFib
cells, passage 12. (B) G-band karyotype analysis of EF+ cells from experiment 1, passage
16. (C) G-band karyotype analysis of EF+ cells from experiment 2, passage 15. (D) The
presence of tetraploid cells in experiment 2 was confirmed using interphase FISH with
two different centromere probes. (E) G-band karyotype analysis of EF+ cells from
experiment 1, passage 35. (F) G-band karyotype analysis of EF+ cells from experiment 2,
passage 37.
Supplemental Figure S5. Dose-response curves of EFFib, EF+, A673, EW8, U2OS and
BJ-fibroblast cells treated with (A) YK-4-279, (B) mithramycin and (C) LY294002 for
three days. Cell viability was measured using CellTiter-Glo luminescence. Data were logtransformed and normalized. Error bars indicate the standard error of the mean of three
experiments.
Supplemental Figure S6. EF+ cells exhibit an Ewing sarcoma gene expression signature.
Gene set enrichment analysis shows enrichment of the Kauer et al. upregulated and
downregulated gene sets (GS1) in the gene expression data from the EF+ versus EF-
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comparison (A) and EF+ versus EFFib comparison (B). The normalized enrichment scores
(NES) and FDR q-values are shown.
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LIST OF SUPPLEMENTAL EXCEL TABLES
Supplemental Table S3. Comparison of gene expression between EF+ and EF- cells
shows 659 genes with differential expression (Fold > 3 and FDR < 0.01).
Supplemental Table S4. Comparison of gene expression between EF+ and EFFib cells
shows 1175 genes with differential expression (Fold > 3 and FDR < 0.01).
Supplemental Table S5. Enrichment of the EF+ versus EFFib upregulated gene signature
in different cell lines in the Cancer Cell Line Encyclopedia. Enrichment was assessed
using the Enrichr tool.
Supplemental Table S6. GSEA was performed with the EF+ versus EF- comparison
using the C2 and C5 gene sets from the MSigDB collection. Gene sets with an FDR qvalue < 0.1 are listed.
Supplemental Table S7. GSEA was performed with the EF+ versus EFFib comparison
using the C2 and C5 gene sets from the MSigDB collection. Gene sets with an FDR qvalue < 0.1 are listed.
Supplemental Table S8. Cell lines from the Cancer Cell Line Encyclopedia (CCLE,
Broad Institute) used in the GSEA analysis.
Supplemental Table S9. EF Overlap gene list. The gene list from EF+ versus EF- was
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compared to the gene list from EF+ versus EFFib to identify an overlapping set of genes
with differential expression (upregulated in red and downregulated in blue).
Supplemental Table S10. Genes that are included on both the EF Overlap list and the
Hancock et al. gene list.
Supplemental Table S11. EF Unique gene list. Genes from the EF Overlap list that are
excluded from the Hancock et al. gene list.
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