Sugihara et al
Supplementary Information
Supplementary Methods
Retroviral infection and bone marrow transplant assay
For the induction of pre-B ALL/LBL from immature cells, bone marrow (BM) cells
were collected by flushing femurs and tibias from 6-8 week old wild-type mice
(C57BL/6) 2 days after intraperitoneal (i.p.) injection of 150 mg/kg 5-fluorouracil
(5-FU; Kyowa Hakko Kirin, Tokyo, Japan).
BM-MNCs were isolated by
lympholyte-M (Cederlane, Uden, Netherlands) and then cultured for 2 days in -MEM
(Invitrogen, San Diego, CA) supplemented with 20% FCS, 100 U/ml penicillin, 100
U/ml streptomycin, 55 mM 2-Mercaptethanol, 100 ng/ml SCF, and 50 ng/ml IL-6 (both
from Peprotech, London, UK). For the induction of pre-B ALL/LBL from committed
B progenitor cells, BM cells were collected from 6-8-week-old wild-type (WT) mice or
Ink4a/Arf–/– mice.
BM-MNCs were isolated as described above and then cultured in
-MEM supplemented with 20% of FCS, 100 U/ml penicillin, 100 U/ml streptomycin,
100 ng/ml SCF and 25 ng/ml IL-7 (Peprotech) for 2 days.
Retroviral infection was
carried out in 35-mm dishes coated with RetroNectin (Takara Bio, Tokyo, Japan)
according to the manufacturer's protocol.
After 3 days of infection, cells were
collected with cell dissociation buffer (Invitrogen) and then used for transplantation, or
subjected to cell sorting to separate HSCs, CLPs (Common lymphoid progenitors), MPs
(Myeloid progenitors) and progenitor B cells. Infected cells (1x10 6 cells) or sorted
cells along with 2x105 WT BM-MNCs, were injected intravenously into lethally X-ray
irradiated (9 Gy) WT mice (C57BL/6).
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Cell sorting
For sorting HSCs, CLPs and MPs, N-Myc-transduced BM cells were treated with a
biotinylated lineage antibody cocktail containing anti-CD5, -B220, -Mac1, -Gr1, -7-4
and -Ter-119 antibodies (Miltenyi Biotech, Bergisch Gladbach, Germany).
Lineage
(Lin)-positive cells were depleted with anti-biotin MicroBeads (Miltenyi Biotech) using
Auto MACS (Miltenyi Biotech).
The remaining cells were stained with
PE-conjugated anti-IL7R(eBioscience, San Diego, CA)APC-conjugated anti-c-kit,
and PE-Cy7-conjugated anti-Sca-1 antibodies (both from Biolegend, San Diego, CA).
Biotinylated antibodies were detected with streptavidin-PE-Texas Red (Biolegend).
For sorting of progenitor B cells (B220+, CD19+, IgD–, IgM–), N-Myc-transduced BM
cells were incubated with anti-B220 antibody conjugated to MicroBeads (Miltenyi
Biotech).
B220+ cells were positively sorted by Auto MACS. Sorted cells were
further stained with PE-conjugated anti-IgD (Biolegend) and -IgM and APC-conjugated
anti-CD19 antibodies.
Cell sorting was performed using FACSVantage SE (BD
Biosciences). All flow cytometry data was analyzed with FlowJo software (Tree Star,
Ashland, OR).
Primary culture of tumor cells
BM-MNCs from tumor-bearing mice in which tumors were derived from WT HSCs or
Ink4a/Arf–/– progenitor B cells were cultured in -MEM (Invitrogen) supplemented with
20% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 ng/ml IL-6. Cells were
maintained at 37˚C in 5% CO2 and 100% humidity.
Apoptosis assay and BrdU incorporation assay
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The cells were washed with cold PBS, resuspended in Annexin V binding buffer (BD
Pharmingen), and then treated with APC-conjugated Annexin V (BD Pharmingen) and
propidium iodide (PI) (Sigma).
For the BrdU incorporation assay, tumor cells were
incubated in -MEM supplemented with 20% FCS and 50 ng/ml IL-6 and containing 10
M BrdU at 37°C for 30 min.
After washing with PBS, cells were fixed,
permeabilized, and then treated with DNase using a BrdU Flow Kit (BD Bioscience).
Cells were stained with APC-conjugated anti-BrdU antibody (BD Bioscience) and
7AAD.
For the apoptosis and BrdU incorporation assays, samples were analyzed
using FACSCalibur.
Quantitative gene expression analysis
Total RNA was extracted from sorted cells using the Trizol reagent (Invitrogen)
according to the manufacturer’s protocol.
Isolated RNA was subjected to
reverse-transcription using a PrimeScript RT-PCR Kit (Takara Bio).
Quantitative
real-time PCR with SYBR Green was performed using the Thermal Cycler Dice Real
Time System (Takara Bio). The PCR conditions were as follows: 95˚C for 2 min,
followed by 40 cycles of 95˚C for 30 s and 60˚C for 30 s, and then dissociation-curve
analysis to confirm specificity.
Data represent means ± SD of triplicate samples.
GAPDH mRNA expression was measured and used to normalize expression.
sequences
of
the
primer
sets
were
5’-CGTACCCCGATTCAGGTGAT-3’
as
follows:
mouse
Ink4a
and
5’-TTGAGCAGAAGAGCTGCTACGT-3’;
5’-GCCGCACCGGAATCCT-3’
mouse
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human
forward,
reverse,
Arf
and
5’-TTGAGCAGAAGAGCTGCTACGT-3’;
The
forward,
reverse,
Ink4a
forward,
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5’-AGCATGGAGCCTTCGGCTGA-3’
and
5’-CCATCATCATGACCTGGATCG-3’;
human
reverse,
Arf
forward,
5’-TACTGAGGAGCCAGCGTCTA-3’
and
reverse,
5’-TGCACGGGTCGGGTGAGAGT-3’;
Bmi1
forward,
and
reverse,
5’-TGCTCGAAAGCCCAATGAAAG-3’
5’-CATAAGTGCCTCAAACGCACTCTC-3’;
mouse
5’-TTCACCACCATGGAGAAGGC-3’
GAPDH
and
5’-CCCTTTTGGCTCCACCCT-3’;
human
5’-GCACCGTCAAGGCTGAGAAC-3’
forward,
reverse,
GAPDH
forward,
and
reverse,
5’-TGGTGAAGACGCCAGTGGA-3’.
Mutational analysis of p53
Total RNA extraction from mouse tumor cells and human B-ALL cell lines and cDNA
synthesis were performed as described above.
amplified
by
PCR
using
the
The entire coding region of p53 was
following
5’-GTGTCACGCTTCTCCGAAGA-3’
primers:
and
mouse
5’-GTCAGTCTGAGTCAGGCC-3’;
human
p53
TCCGGGGACACTTTGCGTT
and
-3’
GGTGCTTCTGACGCACACCT -3’.
human
mouse
p53
p53
forward,
reverse,
forward,
p53
reverse,
5’5’-
PCR products were directly sequenced by
Fasmac (Kanagawa, Japan).
RNA interference
The introduction of short hairpin RNAs (shRNAs) into cells by the retroviral vector
pRePS was performed as previously described (Shimizu et al 2010).
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The sequences of
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the sense oligonucleotides were 5′-GTACATGTGTAATAGCTCC-3′ for p53 shRNA and
5′-CGTACGCGGAATACTTCGA-3′ for luciferase shRNA (nonspecific control).
Histological analysis
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into
4-m thick sections. Sections were depleted of paraffin, rehydrated in a graded series
of ethanol solutions, and then stained with hematoxylin and eosin (H&E).
Peripheral
blood smears were fixed with methanol and then stained with May-Grunwald solution
(Muto Pure Chemicals, Japan) and Giemsa solution (MERCK, Germany).
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Supplementary Figure Legends
Supplementary Figure 1.
(a), The retroviral vector pMXs-IRES-EGFP (pMXs-IG)
was used to express N-Myc, a deletion variant of N-Myc in which Myc-BOX II (MBII)
was deleted and c-Myc (left).
Mouse c-Myc cDNA was kindly provided by Dr. Michel
D. Cole (Dartmouth Medical School).
The empty vector was used as a control.
Protein expression of N-Myc, MBII and c-Myc in infected NIH3T3 cells was
confirmed by immunoblot (right).
control.
-Tubulin was measured in parallel as a loading
(b), Schematic of the BM transplant assay.
BM-MNCs were harvested from
5-FU-treated C57BL/6 mice, pre-stimulated with SCF and IL-6 for 2 days, and then
infected with retroviral vectors for 3 days.
The infected cells were intravenously
transplanted into lethally irradiated syngenic mice.
(c), Survival curve of mice
transplanted with c-Myc-transduced BM-MNCs (n=8).
(d), Representative flow
cytometric immunophenotyping of GFP+ BM cells from a tumor-bearing mouse
transplanted with c-Myc-transduced BM-MNCs.
Supplementary Figure 2.
(a), Hematopoietic stem cell differentiation and
commitment to the B cell lineage.
(b), Representative flow cytometric
immunophenotyping of GFP+ BM-MNCs from tumor-bearing mice transplanted with
N-Myc-transduced CLPs (upper), and histopathology of BM, LN and liver stained with
H&E (lower).
Arrows indicate invaded tumor cells.
Scale bar = 50 m in tissues.
(c), Flow cytometric immunophenotyping of GFP+ BM-MNCs from 2 tumor clones
derived from mice transplanted with N-Myc-transduced MPs.
anti-CD3 antibody was purchased from eBioscience.
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The PE-conjugated
(d), Subpopulations of HSCs,
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CLPs and MPs infected with control (gray) or N-Myc (solid) retroviral vectors in Lin－
GFP+ BM cells
Supplementary Figure 3.
(a), Representative flow cytometric immunophenotyping
of GFP+ BM-MNCs from tumor-bearing mice transplanted with c-Myc-transduced
Ink4a/Arf － / － progenitor B cells.
(b), Percent of survival tumor cells (Annexin
V-negative cells) derived from c-Myc-transduced Ink4a/Arf－/－ progenitor B cells 48 h
after Ara-C treatment is shown in the graph.
Data represent means of triplicates ± SD.
(c), Percent of Annexin V-positive tumor cells derived from c-Myc-transduced Ink4a/Arf
－/ －
progenitor B cells 48 after DMSO or Nutlin-3 (5 M) treatment.
Data represent
means of triplicates ± SD.
Supplementary Figure 4.
(a), p53 mutation sites in tumor cells derived from WT
HSCs and WT Bulk BM cells.
(b), Results of an Annexin V assay of cultured tumor
cells derived from WT (Ink4a/Arf+/+) HSCs and WT Bulk BM cells 48 h after treatment
with Nutlin-3 (2.5, 5 or 10 M) or DMSO (vehicle) as a control.
(c), Percent of
survival cells of each human B-ALL cell line 48 after Ara-C treatment is shown in the
graph.
(d), mRNA expression levels of Arf and Ink4a were determined by quantitative
real-time RT-PCR in N-Myc-transduced HSCs (N-Myc HSC) and progenitor B cells
(N-Myc pro-B), and tumor cells derived from WT HSC (tumor HSC1 and 2) and
Ink4a/Arf－/－ progenitor B cells (tumor Ink4a/Arf－/－). Data represent means ± SD of
triplicate measurements.
Reference
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Sugihara et al
Shimizu T, Ishikawa T, Sugihara E, Kuninaka S, Miyamoto T, Mabuchi Y et al (2010).
c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells
into osteosarcoma accompanied by loss of adipogenesis. Oncogene.
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