Mice
We designed the targeting vector in such a way that the first exon was flanked by two LoxP sites and
an Frt flanked neo cassette (Fig. S1A). Targeted BA1 (C57BL/6 × 129/SvEv) hybrid
embryonic stem cells harboring the targeted Adam10 allele were identified by Southern blot analysis
(Fig. S1Bi). Chimeric mice were generated by C57BL/6 blastocyst injection, and
bred with C57BL/6 mice to generate heterozygous mice. The mice were mated with Flp-transgenic
mice to remove the neo cassette and to generate a floxed Adam10 allele. Those mice were further
mated to generate mice homozygous for the floxed Adam10 allele (Adam10flox/flox). Adam10flox/flox
mice were viable and sterile with no apparent defects (data not shown) and were used as control
animals for the experiments (henceforth referred to as Control mice). Adam10flox/flox mice were
crossed with Mx1-Cre transgenic mice21 to generate inducible conditional ADAM10-knockout mice
(Adam10flox/flox/Mx1-Cre+, henceforth referred to as Adam10/Mx1 mice). Conditional excision of the
floxed allele was achieved by intraperitoneal injection of 250 µg of polyinosinic/polycytidylic acid
(pIpC; Sigma) three times at 2-day intervals. The MPD phenotype became obvious at around 2
weeks after the pIpC treatment and gradually worsened thereafter (data not shown). Therefore, we
analyzed the mice at 4 weeks after the injection unless otherwise described. Excision of the floxed
allele and nearly complete absence of ADAM10 protein in BM cells were confirmed by PCR and
western blot analysis, respectively (Fig. S1Bii,iii). Adam10/Mx1 mice were further
mated with Gcsfr-/- mice24 to generate Adam10/Mx1-Gcsfr-/- double-mutant mice. All animal
experiments were approved by the Institutional Animal Care and Use Committee of School of
Medicine, Keio University.
Flow cytometry
BM cells were isolated by flushing the femurs with RPMI medium. Splenocytes and thymocytes
were collected by mechanical disruption of the tissue samples. Red blood cells were lysed with Red
Blood Cell Lysis Buffer (Roche). The cells were filtered through a cell strainer (BD Falcon) to
remove debris, and preincubated with an anti-CD16/32 antibody (2.4G2; BD Biosciences) to block
nonspecific binding. For the flow cytometric analysis, we used the following
fluorochrome-conjugated monoclonal antibodies: anti-CD3e (145-2C11), anti-CD4 (RM4-5),
anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-B220 (RA3-6B2), anti-Gr-1 (RB6-8C5), anti-Ly-6C
(HK1.4), anti-Ter119 (TER-119), anti-Sca-1 (D7) and c-Kit (2B8). The antibodies were purchased
from Biolegend. A mixture of monoclonal antibodies against CD4, CD8, B220, TER-119, CD11b
and Gr-1 was used as a lineage marker (Lin). The flow cytometric analysis was performed using a
FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (Tree Star Inc.). Dead cells
were excluded from the analysis by propidium iodide staining.
Colony-forming assay
Colony-forming assays were performed using methylcellulose-based medium (Methocult #03534;
StemCell Technologies) as instructed by the manufacturer. BM cells and splenocytes isolated from
8–10-week-old mice pretreated with pIpC were incubated for 7 days and the numbers of colonies
were counted under a light microscope.
Quantitative RT-PCR
Total RNA from the spleen was extracted using RNAiso (Takara Bio, Shiga, Japan) and was
reverse-transcribed using ReverTra Ace (Toyobo, Osaka, Japan). PCR amplification and
quantification were performed using SYBR premix EXtaqII (Takara Bio) and LightCyclerII (Roche).
Relative transcript expression levels were obtained by normalizing to the β-Actin expression.
BM cell transfer
Donor BM cells were collected from Adam10/Mx1 mice (at 1 week after pIpC treatment) or Control
mice. The cells were injected into lethally-irradiated (10.5 Gy) recipient mice via the retro-orbital
vein under general anesthesia. The mice were maintained for 2–3 months before analysis.
Cytokine protein array and determination of cytokine levels
Serum samples were collected from three Adam10/Mx1 mice (at 4 weeks after pIpC treatment) or
Control mice and pooled for the assay. The cytokine levels in the serum samples were evaluated
using a Proteome Profiler Array (Mouse Cytokine Array Panel A; R&D Systems) according to the
manufacturer’s instructions. The serum level of G-CSF was evaluated by a sandwich ELISA
(Quantikine; R&D Systems) according to the manufacturer’s instructions.
Statistical analysis
A t-test for two samples assuming equal variances was used to evaluate the significance of
differences between values. Values of p<0.05 were considered to indicate statistical significance. All
experiments were performed independently at least three times with similar results.
Table S1. Summary of the hematopoietic phenotypes of Notch-signaling defective mutant mice with MPD
Mice
Ps1+/-Ps2-/Mib-1 flox/flox/Mx1-Cre
Mib-1 flox/flox/MMTV-Cre
N1flox/flox/N2flox/flox/K5-CreERT
Rbp-j flox/flox/K5-CreERT
FX-/Ncstn
flox/flox
(N1
Serum
G-CSF
T-Cell
development
+
N.A.
Normal
+
N.A.†
N.A.
+
Elevated
+
(cell-autonomous)
Transferable MPD
-*
Non-cell-autonomous
MPD
etc
Ref
N.A.
18
-
+
17
N.A.
-
+
Serum TSLP↑
19
N.A.
N.A.
+
+
KSL cells→
20
+
N.A.
Defective
+
-
KSL cells↑
25
+
Elevated
Defective
+‡
+
/Mx1-Cre
Ncstn flox/flox/Vav-Cre
flox/flox
MPD
/N2
flox/flox
-/-
/N3 Mx1-Cre)
Adam10 flox/flox/Mx1-Cre
Ps indicates Presenilin; Mib-1, Mind bomb-1; N, Notch; Ncstn, Nicastrin; TSLP, thymic stromal lymphopoetin, and N.A., data not available.
* Commented in Ref (17) as a personal communication.
† No change in the transcript level of G-CSF in the BM and BM stromal cDNAs.
‡ Contribution of cell-autonomous MPD was minor compared to that of non-cell-autonomous MPD.
§ Data not shown.
KSL cells↑
Serum TSLP↑§
Present
study
Figure S1. Generation of Adam10flox/flox mice. (A) Schema for the conditional gene targeting of
Adam10. The arrowheads indicate the locations of the primers used for genotyping. (B) Southern
blot analysis (i) of genomic DNA isolated from gene-targeted embryonic stem cells. W, wild-type
allele; T, targeted allele. PCR analysis (ii) of genomic DNA isolated from Adam10w/f (Ctrl) and
Adam10/Mx1 (A10/Mx1) mice. Fl, Wt and Null indicate the PCR products of the floxed, wild-type
and null alleles, respectively. Western blot analysis (iii) of ADAM10 (A10) and β-Actin (βA) in BM
cells from Adam10flox/flox (Ctrl) and Adam10/Mx1 (A10/Mx1) mice.
Figure S2. Impaired T-cell development in Adam10/Mx1 mice. (A) Gross morphologies of the
thymus from Control and Adam10/Mx1 mice treated with pIpC at 4 weeks prior to euthanasia. (B)
Thymus/body weight ratios and total cell counts of the thymus. Bars indicate means ± SD (n=3). (C)
Representative flow cytometric analysis of thymocytes from Control and Adam10/Mx1 mice. (D)
Absolute cell numbers of CD4-CD8-, CD4+CD8+, CD4+ and CD8+ cells in the thymus of Control and
Adam10/Mx1 mice. **p<0.005.
Figure 3. Multiplex cytokine analysis of serum samples from Control and Adam10/Mx1 mice.
Representative photographs of cytokine arrays using serum samples from pIpC-treated Control and
Adam10/Mx1 mice. Colored boxes indicate differentially expressed cytokines. The serum samples
from three mice were pooled for each experiment.
Figure S1
A
9.6 Kbp
B
B
Wildtype allele
0.17 Kbp
B
6.1 Kbp
B
Targeted allele
B
Neo
probe
Floxed allele
0.45 Kbp
Null allele
0.3 Kbp
1st exon
B
i
Frt
ii
LoxP
B, BamHI
iii
W/W W/T
Ctrl A10/Mx1
Ctrl A10/Mx1
Cre
A10
9.6
Fl
0.45
Wt
0.17
βA
100
75
50
37
6.1
Kbp
Southern blot
Null
0.3
Kbp
genomic PCR
kDa
Western blot
Figure S2
A
B
Adam10/Mx1
absolute number (×107)
**
thymus / body ratio
Control
Control
Adam10/Mx1
0.002
0.0015
0.001
0.0005
0
**
12
8
4
0
12
Control
Adam10/Mx1
10
8
6
4
**
2
8-
8+
0
D
4+
C
D
D
22.5% 4.16%
**
14
C
62.7%
4C
2.62% 2.72%
10.2%
D
CD4
CD8
Adam10/Mx1
86.2%
C
Control
8.67%
absolute number (×107)
D
C
CD4 +
**
CD8 +
Figure S3
Control
Adam10/Mx1
CXCL13
G-CSF
IL-16
TREM1