We designed the targeting vector in such a way that the first exon was flanked by two LoxP sites and

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1 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 (Adam10 flox/flox ). Adam10 flox/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). Adam10 flox/flox mice were crossed with Mx1-Cre transgenic mice 21 to generate inducible conditional ADAM10-knockout mice (Adam10 flox/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 -/- mice 24 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.

2 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).

3 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.

4 Table S1. Summary of the hematopoietic phenotypes of Notch-signaling defective mutant mice with MPD Mice MPD Serum G-CSF T-Cell development (cell-autonomous) Transferable MPD Non-cell-autonomous MPD Ps1 +/- Ps2 -/- + N.A. Normal - * N.A. 18 etc Ref Mib-1 flox/flox /Mx1-Cre Mib-1 flox/flox /MMTV-Cre + N.A. N.A N1 flox/flox /N2 flox/flox /K5-Cre ERT Rbp-j flox/flox /K5-Cre ERT + Elevated N.A. - + Serum TSLP 19 FX -/- + N.A. N.A. + + KSL cells 20 Ncstn flox/flox /Mx1-Cre Ncstn flox/flox /Vav-Cre (N1 flox/flox /N2 flox/flox /N3 -/- Mx1-Cre) + N.A. Defective + - KSL cells 25 Adam10 flox/flox /Mx1-Cre + Elevated Defective + + KSL cells Serum TSLP Present study 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.

5 Figure S1. Generation of Adam10 flox/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 Adam10 w/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 Adam10 flox/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< 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.

6 Figure S1 A 9.6 Kbp B B Wildtype allele 0.17 Kbp 6.1 Kbp B B B Targeted allele Neo Floxed allele Null allele 0.45 Kbp probe 0.3 Kbp 1st exon Frt LoxP B, BamHI B i ii iii W/W W/T Ctrl A10/Mx1 Ctrl A10/Mx1 Cre 9.6 Fl Wt A10 βa Null Kbp Kbp kda Southern blot genomic PCR Western blot

7 Figure S2 A Control Adam10/Mx1 B thymus / body ratio Control Adam10/Mx1 absolute number ( 10 7 ) C D CD4 Control Adam10/Mx1 8.67% 86.2% 10.2% 62.7% 2.62% 2.72% 22.5% 4.16% CD8 absolute number ( 10 7 ) CD4 - CD8 - CD4 + CD8 + Control Adam10/Mx1 CD4 + CD8 +

8 Figure S3 Control Adam10/Mx1 CXCL13 G-CSF IL-16 TREM1