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Cell Stem Cell, Volume 19

Supplemental Information

p38a Activates Purine Metabolism

to Initiate Hematopoietic Stem/Progenitor

Cell Cycling in Response to Stress

Daiki Karigane, Hiroshi Kobayashi, Takayuki Morikawa, Yukako Ootomo, MashitoSakai, Go Nagamatsu, Yoshiaki Kubota, Nobuhito Goda, Michihiro Matsumoto, Emi K.Nishimura, Tomoyoshi Soga, Kinya Otsu, Makoto Suematsu, Shinichiro Okamoto, ToshioSuda, and Keiyo Takubo

Figure S1, related to Figure 1

(A) Expression of mRNAs encoding p38MAPK family proteins (p38α, p38β, p38γ, and p38δ) based on

qPCR of hematopoietic cell fractions, including LT-HSCs (CD34-Flt3-LSK), ST-HSCs (CD34+Flt3-LSK),

MPPs (CD34+Flt3+LSK), myeloid progenitors (Lineage-c-Kit+Sca-1-, LKS-), and lineage-marker-positive

cells (Lin+) (means ± SD, n = 4).

(B) qPCR quantification of p38MAPK family transcripts (p38β, p38γ, and p38δ) in LT-HSCs, plus

corresponding positive controls (means ± SD, n = 4).

(C) Quantification of Mapk14 deletion efficiency in p38α+/+ or p38αΔ/Δ PBs (means ± SD, n = 10), MPPs

(CD34+Flt3+LSK), ST-HSCs (CD34+Flt3-LSK) and LT-HSCs (CD34-Flt3-LSK) (means ± SD, n = 4) by

genomic qPCR. Exon 2 is targeted for deletion, while exon 12 is outside the deletion site.

(D) Quantification of Mapk14 deletion efficiency by genomic qPCR in p38α+/+ or p38αΔ/Δ PB cells (means

± SD, n = 3) following tamoxifen injection. Exon 2 is targeted for deletion, while exon 12 is outside the

deletion site.

(E) qPCR quantification of p38MAPK family transcripts (p38β, p38γ, and p38δ) among HSPCs (LT-HSC

(CD34-Flt3-LSK), ST-HSCs (CD34+Flt3-LSK) and MPPs (CD34+Flt3+LSK) from p38α+/+ or p38αΔ/Δ mice.

(means ± SD, n = 4).

(F-J) Analysis of hematological phenotypes seen in p38αΔ/Δ mice. PB counts (white blood cell (WBC), red

blood cell (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean

corpuscular hemoglobin (MCH) and platelet (Plt)) (F), PB differentiation status (B220+ B cells (B), CD4+

T cells (CD4), CD8+ T cells (CD8), Mac-1+Gr-1hi granulocytes (Gra) or Mac-1+Gr-1lo macrophages (Mac))

(G), frequencies of BM HSPCs (common lymphoid progenitors (CLP; Lin-IL7Rα+Flt3+Sca-1/c-Kitlo),

megakaryocyte-erythroid progenitors (MEP; Lin-IL7Rα-Sca-1-c-Kit+CD16/32-CD34-), granulocyte-

monocyte progenitors (GMP; Lin-IL7Rα-Sca-1-c-Kit+CD16/32+CD34+), common myeloid progenitors

(CMP; Lin-IL7Rα-Sca-1-c-Kit+CD16/32-CD34+), MPPs (CD34+Flt3+LSK), ST-HSCs (ST; CD34+Flt3-LSK),

LT-HSCs (LT; CD34-Flt3-LSK) or SLAM LSK cells (SLAM; CD41/48-CD150+LSK) (H), frequencies of

spleen HSPCs, including CLPs (common lymphoid progenitors; Lin-IL7Rα+Flt3+Sca-1/c-Kitlo), MEPs

(megakaryocyte-erythroid progenitors; Lin-IL7Rα-Sca-1-c-Kit+CD16/32-CD34-), GMPs (granulocyte-

monocyte progenitors; Lin-IL7Rα-Sca-1-c-Kit+CD16/32+CD34+), CMPs (common myeloid progenitors; Lin-

IL7Rα-Sca-1-c-Kit+CD16/32-CD34+), MPPs (CD34+Flt3+LSK), ST-HSCs (CD34+Flt3-LSK) or LT-HSCs

(CD34-Flt3-LSK) (I), and frequencies of thymic T cell fractions including double negative (DN; CD4-CD8-),

double positive (DP; CD4+CD8+), CD4+CD8- (CD4 SP) and CD4-CD8+ (CD8 SP) in 10-week-old p38α+/+

or p38αΔ/Δ mice (means ± SD, n = 4) (J).

(K) Quantification of Mapk14 deletion efficiency by genomic qPCR in p38α+/+ or p38αΔ/Δ PB cells (means

± SD, n = 5), MPPs (CD34+Flt3+LSK), ST-HSCs (CD34+Flt3-LSK) and LT-HSCs (CD34-Flt3-LSK) (means

± SD, n = 4) 12 weeks after transplantation. Exon 2 is targeted for deletion; exon 12 is outside the deletion

site.

(L) Limiting dilution analysis using BMMNCs. Three different doses of p38α+/+ or p38αΔ/Δ BMMNCs (1 x

104, 2 x 104 and 3 x 104) were transplanted into lethally-irradiated recipient mice together with 4 x 105

competitor BMMNCs. Recipients were considered reconstituted if the percentage of donor-derived cells

in PB was no less than 5% at 3 months post transplantation. Frequencies seen in non-reconstituted mice

were plotted against dose of transplanted LT-HSCs. The frequency of functional HSCs was calculated

using ELDA software (http://bioinf.wehi.edu.au/software/elda/index.html).

(M) Expression of mRNAs encoding p38MAPK family members (p38α, p38β, p38γ, and p38δ) based on

qPCR of bone marrow niche fractions, including mesenchymal stromal cells (MSC; CD45-Ter119-CD31-

CD140a+LepR+), bone marrow endothelial cells (EC; Ter119-CD45-CD31+), and leptin receptor-negative

cells (LepR-; Ter119-CD45-CD31-CD140a-LepR-) (means ± SD, n = 4).

(N) Chimerism of donor-derived PB cells following primary BMT (means ± SD, n = 4-5). BMMNCs (Ly5.1)

were transplanted into lethally-irradiated p38α+/+ or p38αΔ/Δ recipients. Percentage of donor-derived cells

among PB cells of Ly5.1+ cells at indicated months after BMT are shown.

(O) Chimerism of donor-derived BM cells including LT-HSCs (CD34-Flt3-LSK), ST-HSCs (CD34+Flt3-LSK),

MPPs (CD34+Flt3+LSK), and LKS- (Lineage-c-Kit+Sca-1-) and Lin+ (lineage marker-positive) cells from

recipients 4 months after BMT (means ± SD, n = 4-5).

*P < 0.05, **P < 0.01.


(A) Analysis of PB cell counts (white blood cell (WBC; 1 x 102 /μl), hemoglobin (HGB; g/dl) and platelet

(PLT; 1 x 104 /μl)) on days 0, 3 and 6 following 5-FU injection (means ± SD, n = 5).

(B) p38MAPK phosphorylation levels decrease relative to controls in p38αΔ/Δ lineage-negative BM cells

by day 3 after 5-FU injection. Mean fluorescence intensity (MFI) was analyzed by intracellular flow

cytometry. Representative histograms of p38MAPK phosphorylation in p38α+/+ (left) and p38αΔ/Δ (right)

cells and actual MFI are shown.

(C) Representative flow cytometry gating strategies used to identify BM hematopoietic cells on days 3

and 6 after 5-FU injection.

(D) Representative flow cytometry gating strategies used to identify BM hematopoietic cells from pre-

BMT to day 7 after BMT.

(E) Representative histograms showing p38MAPK phosphorylation (red) and isotype controls (gray) in

LSK cells from pre-BMT to day 7 after BMT.


(A) Number of donor-derived HSPCs including LT-HSCs (CD34-Flt3-LSK), ST-HSCs (CD34+Flt3-LSK)

and MPPs (CD34+Flt3+LSK) 16 hours after BMT (mean ± SD, n = 10).

(B) Chimerism of donor-derived PB cells following BMT (means ± SD, n = 9). LT-HSCs (CD34-Flt3-LSK)

from p38α+/+ or p38αΔ/Δ mice were transplanted intrafemorally into lethally-irradiated recipients together

with competitor BMMNCs. Percentages of donor-derived cells among PB cells of p38α+/+ or p38αΔ/Δ mice

at indicated months after BMT are shown.

(C) Chimerism of donor-derived LT-HSCs (CD34-Flt3-LSK) in each leg bone 4 months after intrafemoral

BMT (means ± SD, n = 8-9). LT-HSCs from p38α+/+ or p38αΔ/Δ mice were transplanted into the left femur

of lethally-irradiated recipients.

(D) Immunohistochemical analysis of CFSE (green)-labeled p38α+/+ or p38αΔ/Δ transplanted LT-HSCs

(CD34-Flt3-LSK). BM sections were stained with DAPI (gray) and anti-PLVAP (blue). Lodged CFSE-

positive LT-HSCs were identified by their location outside the PLVAP-positive BM vasculature based on

CFSE and DAPI fluorescence.

Scale bar, 20 μm; arrowhead, CFSE-positive transplanted LT-HSCs. Quantification (left) and

representative confocal images (right) of lodged LT-HSCs are shown.

(E) Annexin V-positive apoptotic donor-derived LT-HSCs (CD34-Flt3-LSK) 16 hours after BMT (means ±

SD, n = 4-6).

(F) Redox-sensitive MitoTracker Orange CMH2TMROS fluorescence in donor-derived LT-HSCs (CD34-

Flt3-LSK) 16 hours after BMT (means ± SD, n = 4-6).

(G) Cell division analysis of transplanted HSPCs, including LT-HSCs (CD34-Flt3-LSK), ST-HSCs

(CD34+Flt3-LSK), MPPs (CD34+Flt3+LSK) and myeloid progenitors (Lineage-c-Kit+Sca-1-, LKS-), based

on CFSE labeling. Fluorescence of CFSE-labeled, transplanted HSPCs was analyzed on days 1, 2 and

3 after BMT (means ± SD, n = 5).

(H) Cell cycle status of indicated HSPC fractions including LT-HSCs (CD34-Flt3-LSK), ST-HSCs

(CD34+Flt3-LSK), and MPPs (CD34+Flt3+LSK) as measured by BrdU staining. BM replacement chimeras

were administered BrdU once, 24 hours before assessment of BrdU incorporation. Percentage of BrdU-

positive cells is shown (means ± SD, n = 5).

*P < 0.05, **P < 0.01.


(A-B) CE-TOFMS-based quantification of anionic (A) or cationic (B) metabolites among p38α+/+ or

p38αΔ/Δ LSK cells at steady state (cells pooled from 3 mice) or 16 hours after BMT (cells pooled from 25

mice).

(C) Expression of Impdh (Impdh1 and Impdh2) and Gmps mRNAs in p38α+/+ or p38αΔ/Δ LT-HSCs (CD34-

Flt3-LSK) at steady state or on days 1 (BMT D1) and 2 (BMT D2) after BMT (means ± SD, n = 4). Values

are normalized to β-actin expression.

(D) CE-TOFMS-based quantification of metabolites in EML-C1 cells cultured with 50 μM SB203580 (SB)

or vehicle (means ± SD, n = 3-5). ΣPPP: gluconate + 6-phophogluconate + Ribulose 5-phosphate +

Ribose 5-phosphate + D-sedoheptulose 7-phosphate + Erythrose 4-phosphate. Energy charge:

(ATP+1/2ADP)/(ATP+ADP+AMP).

*P < 0.05, **P < 0.01.


(A) qPCR analysis of Mitf transcripts in hematopoietic cell fractions, including LT-HSCs (CD34-Flt3-LSK),

ST-HSCs (CD34+Flt3-LSK), MPPs (CD34+Flt3+LSK), myeloid progenitors (Lineage-c-Kit+Sca-1-, LKS-),

and lineage-marker-positive cells (Lin+) (means ± SD, n = 4).

(B) Analysis of hematological phenotypes in Mitfvit/vit mice. Shown is PB differentiation status (B220+ B

cells (B), CD4+ T cells (CD4), CD8+ T cells (CD8), Mac-1+Gr-1hi granulocytes (Gra) or Mac-1+Gr-1lo

macrophages (Mac)) in 10-week-old Mitf+/+ or Mitfvit/vit mice (means ± SD, n = 5).

(C) Cell division analysis of transplanted LT-HSCs (CD34-Flt3-LSK). CFSE fluorescence was analyzed

on days 3 and 7 after BMT (means ± SD, n = 5).

(D) qPCR analysis of Mitf and Impdh2 transcripts in EML-C1 cells treated with 50 μM SB203580 or vehicle

(means ± SD, n = 4).

*P < 0.05, **P < 0.01.


Flow cytometric analysis of LSK cells transduced with pMY-ires-EGFP or pMY-Impdh2-ires-EGFP

retroviral vectors. Representative flow cytometry gating strategies used to identify control or Impdh2

vector-positive p38α+/+ or p38αΔ/Δ LSK cells.

Table S1, related to Figure 3

Genes included in the Gene Set Enrichment Analysis (GSEA) in Figure 3F (“HSC_VS_MPP_UP” gene

set).

GM10883 RYK KRT8 TINAGL1 UNC45B IRF6CAR1 AKR1C19 D0H4S114 CORO2B LHCGR TSPAN8IGJ RARB NUPR1 SFRP1 STOX2 CPLX2NEO1 CD74 PPP1R16B D7ERTD715E MT2 RTKNGM10883 GBP8 PKP2 MT1 GM1337 PTK2MEG3 FSCN1 H2-AA PLK2 TRPC6 LGR4A930038C07RIK EPAS1 VNN1 MUM1L1 FADS3 ADCK3SULT1A1 CDKN1C HOXB5 PDK4 TCF7L1 ITSN1RUNX1T1 PFN2 IKZF4 SLC15A2 TMTC2 PLSCR2ERMAP 1810019J16RIK NRK C230098O21RIK EMR4 HOPXIGH-6 SBF2 KLF1 IGH-2 PRKCB NINLCLU CALML4 PDE2A NDRG4 AK1 SMAGPIGF1 TGM2 KLHL4 D630039A03RIK AMPD3 CARD10PDGFC ART4 D17ERTD663E PRR15 BEST1 SDC4VWF WFDC2 OSGIN1 2010309G21RIK CRYAB MPDZCLCA1 RHOJ DIXDC1 UPP1 ABCB1A IGL-C1GPR64 2810030E01RIK CPNE8 EVC KLRD1 WTIPAHSP MAPK12 PLCL1 MBOAT2 PTPLAD2 SLC6A15GM10419 NBEA PRKD1 PRNP IGF2R BEX4GDA MLLT3 EXPH5 LOC100502594 IL7R C030034E14RIKGBP10 CLDN5 PLAGL1 KRT7 SLC16A2 TULP3GIMAP4 CD38 INHA PROS1 PABPC4L 9330156P08RIKZFP612 FAM55D IL4 MEIS2 1600021P15RIK PLXDC2CLEC1A GNAI1 ENAH FHL1 CBR3 SEMA7APGR ST3GAL6 REPS2 GSTT1 SELM EGR1OBSL1 FAM171B BANK1 FAP SDC2 JAM3FAM46C A730089K16RIK LDHD RNF208 ZG16 RORAH19 CLEC14A SLAMF1 RHBDF1 MYH6 TRIM47SHROOM3 PYGM ALDH1A1 NUDT11 IIGP1 FAM55BSDPR NR3C2 NTF5 TRAF1 FAM110C CTNNAL1OBSCN UNC13B 4832441B07RIK CTSH NCKAP1 BC020535CLCA1 NDN ECSCR POU2AF1 C1RA PREX2AIRN JAM2 CLIP3 AQP1 EHD3 PORCNVPREB1 HBA-A1 MYH10 GM11346 PTPN14 GHRSCRN1 TENC1 CHD7 2010107G23RIK SH3YL1 NKX2-3KAZALD1 TMEM56 FGD5 ACPP CHCHD7 SEMA4FKLHL13 5930427L02RIK APBB1 SLFN2 AHNAK FSTL1IGF2BP2 SPIRE1 PDGFD ZFP37 HIF3A CISHMPA2L CES2G GM5148 EVC2 H2-EB1 GPRIN2LANCL3 SOCS2 ABCG3 IGDCC4 ABCA5 PDZK1IP1TRIB3 2510009E07RIK RIPPLY3 ANKRD33B 9630023C09RIK SQRDL

BC051142


Metabolome analysis using EML-C1 with SB203580 or vehicle.

Glycolysis Mean SD Mean SD Amino acids Mean SD Mean SDGlucose 1-phosphate 125.9 ± 23.4 36.5 ± 4.7 Gly 554.3 ± 183.0 998.2 ± 167.4Glucose 6-phosphate 696.7 ± 106.7 67.5 ± 5.4 Ala 552.3 ± 133.6 399.3 ± 104.3Fructose 6-phosphate 270.5 ± 71.4 19.6 ± 1.5 Ser 186.5 ± 33.3 182.7 ± 32.0Fructose 1,6-bisphosphate 125.5 ± 65.4 154.5 ± 19.2 Thr 1220.5 ± 247.2 611.7 ± 357.0Dihydroxyacetonephosphate 88.8 ± 14.9 19.8 ± 2.2 Val 1142.3 ± 219.9 1416.3 ± 264.12,3-Bisphosphoglycerate 9.2 ± 4.4 31.6 ± 2.1 Ile 517.6 ± 68.1 579.1 ± 155.23-Phosphoglycerate 97.4 ± 36.0 44.8 ± 4.5 Leu 565.3 ± 134.2 630.0 ± 84.5Phosphoenolpyruvate 96.4 ± 22.5 9.4 ± 1.3 Lys 473.2 ± 44.8 469.4 ± 39.9Pyruvate 52.0 ± 30.0 84.7 ± 5.0 Arg 440.1 ± 43.3 524.9 ± 160.6Lactate 5573.0 ± 1147.8 792.7 ± 35.7 His 367.4 ± 40.5 393.0 ± 86.4

Tyr 561.9 ± 310.6 2118.9 ± 2588.3Glycerol phosphate shuttle Phe 404.8 ± 77.0 374.4 ± 118.5Glycerol 3-phosphate 203.0 ± 74.9 89.2 ± 74.1 Trp 75.8 ± 29.8 171.4 ± 127.5

Pro 1346.2 ± 381.0 1397.6 ± 231.4TCA Cycle Gln 1570.4 ± 176.1 1404.4 ± 189.5Acetyl CoA 67.1 ± 17.6 45.6 ± 20.7 Glu 8215.9 ± 2967.4 4093.4 ± 924.3Citrate 273.7 ± 282.7 190.2 ± 213.0 Asn 675.1 ± 67.8 417.9 ± 165.7cis-Aconitate 121.0 ± 38.3 73.5 ± 24.0 Asp 1898.9 ± 266.7 913.2 ± 90.9Isocitrate 42.3 ± 49.3 47.7 ± 47.62-Oxoglutarate 167.5 ± 53.4 53.8 ± 33.0 Nucleic acidsSuccinate 488.7 ± 221.3 228.0 ± 14.8 Adenine 70.6 ± 18.0 33.1 ± 2.7Fumarate 183.3 ± 61.8 114.3 ± 27.0 Guanine 111.9 ± 23.9 74.6 ± 34.5Malate 870.3 ± 305.8 356.4 ± 62.5 Cytosine 127.9 ± 17.4 129.3 ± 17.3

Uracil 218.0 ± 12.5 79.2 ± 99.6Pentose phosphate pathway Adenosine 140.4 ± 26.1 104.8 ± 6.6Gluconate 105.5 ± 47.4 10.4 ± 1.8 Guanosine 112.2 ± 71.0 81.2 ± 78.76-Phosphogluconate 76.2 ± 21.9 39.6 ± 32.0 Cytidine 224.2 ± 61.9 60.4 ± 43.4Ribulose 5-phosphate 70.4 ± 12.8 72.8 ± 28.9 Thymidine 5023.7 ± 571.9 3556.6 ± 284.0Ribose 5-phosphate 65.8 ± 27.6 52.6 ± 26.7 Uridine 94.8 ± 23.4 10.7 ± 6.6D-Sedoheptulose 7-phosphate 241.9 ± 26.0 94.6 ± 68.1 Inosine 126.0 ± 131.4 25.2 ± 6.7Erythrose 4-phosphate 24.3 ± 12.3 34.6 ± 22.9 AMP 2125.1 ± 1067.8 1093.3 ± 180.9

GMP 372.9 ± 194.0 160.6 ± 73.9Nucleic acid synthesis CMP 159.9 ± 122.1 61.4 ± 90.4PRPP 8.1 ± 7.3 4.5 ± 1.6 TMP 25.7 ± 25.1 22.8 ± 6.5

UMP 650.2 ± 415.3 227.6 ± 141.0Methylated products IMP 372.8 ± 226.2 223.2 ± 115.2Met 211.9 ± 75.8 129.7 ± 57.5 cAMP 27.7 ± 44.0 7.6 ± 4.3SAM 392.8 ± 9.3 261.7 ± 35.6 cGMP 48.0 ± 47.0 14.9 ± 8.2Spermidine 35.1 ± 9.3 30.3 ± 10.0 cCMP 8.3 ± 9.4 15.2 ± 13.1Spermine 15.9 ± 8.5 16.8 ± 19.9 cTMP 24.8 ± 27.1 16.5 ± 10.6SAH 30.9 ± 22.0 21.0 ± 1.8 dUMP 15.1 ± 6.4 29.7 ± 23.6Cystathionine 399.5 ± 111.7 252.5 ± 24.6 ADP 3558.0 ± 2142.2 1588.9 ± 395.2Hypotaurine 55.7 ± 25.7 42.2 ± 27.1 GDP 755.6 ± 413.7 329.4 ± 86.0Taurine 1076.6 ± 361.0 818.1 ± 706.0 CDP 243.6 ± 227.6 22.7 ± 30.4Thiotaurine 11.8 ± 4.9 17.9 ± 16.3 TDP 17.0 ± 18.4 9.1 ± 6.1GSH 500.9 ± 131.5 124.1 ± 25.9 UDP 392.5 ± 428.3 127.7 ± 103.6GSSG 62.5 ± 4.7 111.4 ± 13.9 ATP 6134.6 ± 1353.9 3018.5 ± 1249.3N-Methyl-Arg 159.3 ± 29.3 185.1 ± 35.6 GTP 1046.1 ± 270.9 585.4 ± 145.0ADMA 157.9 ± 25.9 150.2 ± 32.0 CTP 444.0 ± 296.2 15.4 ± 6.7SDMA 159.2 ± 23.3 125.9 ± 18.9 TTP 13.9 ± 9.6 41.1 ± 33.2

UTP 1248.8 ± 533.8 502.6 ± 268.4Urea cycle dATP 27.1 ± 22.2 12.2 ± 9.1Ornithine 149.2 ± 45.3 274.2 ± 38.9 dGTP 100.5 ± 157.9 288.8 ± 294.3Citrulline 189.8 ± 16.8 165.4 ± 87.2 dCTP 26.9 ± 33.7 5.5 ± 3.3Creatine 345.2 ± 86.6 581.7 ± 18.0 Hypoxanthine 58.2 ± 32.3 42.0 ± 2.3Creatine phosphate 83.5 ± 26.6 44.8 ± 45.5 Xanthine 169.9 ± 122.8 61.6 ± 36.0Creatinine 310.8 ± 94.0 463.7 ± 34.7 NAD 797.3 ± 573.6 610.6 ± 403.2Hydroxyproline 279.9 ± 66.8 190.6 ± 65.0 NADH 82.9 ± 109.7 29.5 ± 32.7

NADP 70.9 ± 62.0 40.0 ± 8.0NADPH 91.6 ± 42.2 37.0 ± 28.8FAD 12.2 ± 7.9 21.7 ± 16.5

Fatty acid metabolismL-Carnitine 378.4 ± 127.7 645.1 ± 66.5CoA 91.6 ± 60.8 5.2 ± 3.6Malonyl CoA 579.5 ± 240.1 339.7 ± 160.7

Vehicle SB203580pmol / 1 x 107 EML-C1 cells (n=3-5)

Vehicle SB203580pmol / 1 x 107 EML-C1 cells (n=3-5)


Primers for ChIP-qPCR assay in Figure 5F and 5G.

Mitf promoter regionF acagtagaagcccaggctgaR ggatatcagcttccgcaaaa

Impdh2 promoter regionF cttaattgaatgggcctggatccR gtagggcgtgcgtactgagggcc

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Antibodies

The following monoclonal antibodies (mAbs) were used in this study: c-Kit (2B8; eBioscience), Sca-1

(E13-161.7; BioLegend), CD4 (L3T4; BD Biosciences), CD8a (53-6.72; BD Biosciences), B220 (RA3-

6B2; BD Biosciences), TER-119 (BioLegend), Gr-1 (RB6-8C5; BD Biosciences), CD34 (RAM34;

eBioscience), Mac-1 (M1/70; BD Biosciences and eBioscience), Flt-3 (A2F10.1; BioLegend), CD45.2

(104; BD Biosciences), CD45.1 (A20; BD Biosciences), CD16/32 (93; eBioscience), IL7Rα (SB/119;

BioLegend), CD41 (MWReg30; BD Biosciences), CD48 (HM48-1; BioLegend), CD150 (TC15-12F12.2;

BioLegend), CD45 (30-F11; BD Biosciences), CD31 (MEC 13.3; BD Biosciences), CD140a (APA5;

eBioscience), goat anti-Leptin receptor polyclonal antibody (R&D systems) and human CD8a (RPA-T8;

BioLegend). A mixture of mAbs against CD4, CD8, B220, TER-119, Mac-1 and Gr-1 served as a lineage

marker (Lineage). Mouse anti-BrdU-Alexa Fluor 647 antibody (3D4; BD Biosciences) was used to detect

intracellular BrdU, and mouse anti-phosphorylated-p38MAPK-PE antibody (36/p38; BD Biosciences) and

mouse-IgG1-PE (BD Biosciences) was used to detect phosphorylated p38MAPK by intracellular flow

cytometry.

Flow cytometry

Mononuclear cells (MNCs) were isolated by centrifugation and hemolysis of total BM (femur and tibia),

PB, spleen and thymus cells. MNCs were stained with antibodies for surface markers including lineage

markers (described above), CD45.1, CD45.2, stem and progenitor markers for 30 min. The following

mAbs were used in this study as stem and progenitor markers: c-Kit, Sca-1, Flt-3, CD34, CD16/32,

IL7Rα, CD41, CD48 and CD150. Stained cells were analyzed by SORP FACSAria (BD Biosciences).

For intracellular flow cytometry analysis of phosphorylated p38MAPK, cells were first stained with

antibodies for surface markers, fixed in 4% PFA for 10 min, and permeabilized with 90% methanol for

30 min, followed by staining with anti-phospho-p38MAPK antibody. To detect mitochondrial ROS

production, sorted cells were stained with 100 nM Mitotracker Orange CMH2TMROS (MolecularProbes)

for 30 min and analyzed by flow cytometry. Data were analyzed using FlowJoTM software (Tree Star

Inc).

Isolation of mesenchymal and endothelial cells

Isolation of mesenchymal and endothelial cells was performed as described by Zhou et al.(Zhou et al.,

2014) with slight modification. Bone marrow was flushed with ice-cold Hank’s balanced salt solution

(HBSS, Gibco) using a 1 ml syringe. The bone marrow plug was incubated in 1 ml digestion solution

(200 U/ml DNase I (Sigma), 250 mg/ml LiberaseDL (Roche) in HBSS plus Ca2+ and Mg2+) at 37℃ for

10 minutes. After a brief vortex, bone marrow was allowed to sediment for 3 minutes and the

supernatant was transferred to another tube on ice. Undigested bone marrow was subjected to a

second round of digestion. After 2 rounds, undigested bone marrow was suspended by gentle pipetting

and mixed with supernatant from the first round of digestion, followed by filtration through a 100-mm

nylon mesh. Cells were washed by centrifugation in PBS/2% FCS, followed by hemolysis as in

preparation for flow cytometry.

Serum free HSC culture

Sorted cells were cultured on U-bottomed fibronectin-coated plates and maintained in SF-O3 medium

(EIDIA) containing 1.0% BSA, 100 ng/ml mouse stem cell factor (SCF; PeproTech) and 100 ng/ml human

thrombopoietin (TPO; PeproTech) with or without following reagents: SB203580 (SB; Cayman Chemical

Company), mycophenolic acid (MPA; Wako). After 3 or 7 days, cells were collected, stained with

fluorophore-labeled mAb, and analyzed by flow cytometry.

EML-C1 culture

EML-C1 cells (Tsai et al., 1994) were cultured in Iscove’s Modified Dulbecco’s Media (IMDM; Life

technologiesTM) medium containing 20% fetal calf serum (FCS; BioWest) and 100 ng/ml SCF with 50 μM

SB203580 or vehicle.

BM immunohistochemistry

9 x 104 CFSE-labeled LT-HSCs (CD34-Flt3-LSK) from p38αΔ/Δ or p38α+/+ mice were transplanted into

lethally-irradiated (9.5Gy) recipients (Ly5.1). Femoral bones from recipients were sectioned and

immunostained according to the Kawamoto method (Kawamoto, 2003; Yamazaki et al., 2011). BM

sections were stained using an Alexa Fluor 647-conjugated anti-PLVAP antibody (MECA-32; AbD Serotec,

diluted 1:100 in Protein Block (Dako)) and DAPI for 90 minutes at room temperature.

Immunofluorescence data were obtained and analyzed with a confocal laser scanning microscope

(FV1000-D; Olympus) at room temperature. Transplanted cells were counted at 20x magnification.

Lodged CFSE-positive LT-HSCs were identified by their location outside the PLVAP-positive BM

vasculature, based on CFSE and DAPI fluorescence.

Bone marrow transplantation

A total of 500 LT-HSCs (CD34-Flt3-LSK) from p38α+/+ or p38αΔ/Δ mice (Ly5.2) or 4 x 105 BMMNCs from

CAG-CreERT2 (-)-p38αflox/flox or p38αfl/fl mice (Ly5.2), together with 4 x 105 BMMNCs (Ly5.1), were

transplanted into lethally-irradiated (9.5Gy) recipients (Ly5.1). For MPA treatment, 500 LT-HSCs (CD34-

Flt3-LSK) from C57BL/6J mice were cultured in SF-O3 medium containing 1.0% BSA, 100 ng/ml SCF,

and 100 ng/ml TPO with or without MPA and transplanted into lethally-irradiated (9.5Gy) C57BL/6J-Ly5.1

mice. On days 1, 2, 3, and 7 after BMT, CFSE fluorescence or pp38MAPK levels were detected by flow

cytometry. Four months after BMT, 1 x 106 BMMNCs from primary recipients were intravenously

transplanted into lethally-irradiated (9.5Gy) secondary recipients (Ly5.1). For the reciprocal

transplantation method, 1 x 106 BMMNCs (Ly5.1) were transplanted into lethally-irradiated (9.5Gy)

recipients (Ly5.2, p38α+/+ or p38αΔ/Δ). Intra-femoral injection was performed as described (Zhong et al.,

2002). The distal end of femur was surgically exteriorized and a 25G needle inserted into the left femoral

medullary cavity. LT-HSCs (500 cells in 3 μl) were transplanted into that cavity of lethally-irradiated

(9.5Gy) recipients (Ly5.1) using a Hamilton microsyringe, and 4 x 105 BMMNCs (Ly5.1) were transplanted

intravenously. To assess the impact of purine metabolism on proliferation following transplantation, 1 x

107 BMMNCs were transplanted into lethally-irradiated (9.5Gy) recipients. For MPA experiments, donor

cells were pretreated with 10 μg/ml MPA for 3 hours, and recipients were then administered an additional

single injection of 10 μg/ml MPA. For MMF experiments, recipients were administered 300 mg/kg/day

mycophenolate mofetil (MMF; Roche) orally after BMT.

Quantitative RT-PCR

Quantitative PCR was performed as described (Takubo et al., 2008). PCR primers were purchased from

TaKaRa Bio.

cDNA microarray analysis

p38α+/+ or p38αΔ/Δ BMMNCs were transplanted into lethally-irradiated (9.5Gy) recipients (Ly5.1). Donor-

derived LT-HSCs (CD34-Flt3-LSK) and MPPs (CD34+Flt3+LSK) pooled from BM of ten mice per group

were sorted 16 hours after BMT. mRNA was extracted using an RNeasy Mini Kit (Qiagen), and then cDNA

was synthesized and hybridized to the gene chip Mouse 60k (Agilent Technologies) for analysis of gene

expression.

GSEA

Normalized expression data were analyzed using GSEA v2.0.13 software (Broad Institute). Most gene

sets were obtained from the Molecular Signatures Database v4.0 distributed at the GSEA website

(http://www.broadinstitute.org/gsea/index.jsp). We generated some gene sets using Gene Expression

Omnibus (accession: GSE56952) (Seita et al., 2012). The “HSC_VS_MPP_UP” gene set was made up

of the top 247 genes from the list; ratios of average expression in HSCs relative to MPPs were arranged

in descending order (Table S1). The number of permutations was set to 1000. Gene sets with a nominal

p-value of <0.05 and a false rate q-value of <0.25 were considered statistically significant.

ChIP and quantitative PCR

EML-C1 cells were cultured with SB203580 or vehicle for 16 hours and crosslinked with 1% formaldehyde

at room temperature for 15 min, and the reaction was stopped by addition of 200 mM glycine. Cells were

rinsed three times in cold PBS, resuspended in buffer-1 (50 mM Tris-HCl (pH 8.5), 10 mM EDTA (pH 8.0),

1% SDS, NP-40, a protease inhibitor cocktail (PI; cOmpleteTM, SIGMA), and 1 mM phenylmethylsulfonyl

fluoride (PMSF), kept at 4°C on ice for 2 hours, and then DNA was fragmented using a sonicator

(Bioruptor® CP-80R). After centrifugation, the supernatant was diluted with a one tenth volume of buffer-

2 (5.556 mM Tris-HCl (pH 7.6), 166.7 mM NaCl, 0.111% sodium-deoxycholate, 1.111% 1% Triton X-100,

PI and PMSF). 50 μl of Dynabeads Protein G® (Invitrogen) preloaded with 10 μl of each antibody (anti-

CREB (abcam) and anti-Mitf (Cell Signaling Technology)) were added to lysates overnight and then

beads were washed 5 times with buffer (50 mM HEPES-KOH (pH 7.4), 500 mM LiCl, 1 mM EDTA, 1%

NP-40, and 0.5% sodium-deoxycholate). Bound DNA was eluted and quantified by real-time PCR. Table

S3 lists primers used.

Retrovirus transduction

LSK cells from p38α+/+ or p38αΔ/Δ mice were sorted and cultured in serum-free SF-O3 medium

supplemented with 100 ng/mL TPO and 100 ng/mL SCF. LSK cells were transduced, using CombiMag

(OZ Biosciences), with retrovirus carrying Impdh2 in a pMY–internal ribosome entry site (ires)–green

fluorescent protein (GFP) vector or a pMX-ires-human CD8 (hCD8) vector (Nosaka et al., 1999; Yamasaki

et al., 2006). Transduced LSK cells were cultured for 48 hours in serum-free SF-O3 medium

supplemented with 100 ng/mL TPO and 100 ng/mL SCF. GFP+ p38α+/+ or p38αΔ/Δ LSK cells and GFP+

Mitf+/+ or Mitfvit/vit Lin- cells were sorted (Figure S6) and cultured in serum-free SF-O3 medium

supplemented with 100 ng/mL TPO, 100 ng/mL SCF and 10 μM EdU for 2 hours, and then cells were

permeabilized to detect intracellular EdU using a Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit

(Life Technologies) and analyzed by MACSQuant (Miltenyi Biotec). For the CFSE assay in vivo, hCD8+

p38α+/+ or p38αΔ/Δ LSK cells were sorted and stained with CFSE before transplantation. Transplanted

cells were analyzed by flow cytometry. For the competitive bone marrow transplantation, GFP+ p38α+/+

or p38αΔ/Δ CD150+CD48-LSK cells and GFP+ Mitf+/+ or Mitfvit/vit CD150+CD48-LSK cells were sorted, and

1500 CD150+CD48-LSK cells, together with 4 x 105 BMMNCs (Ly5.1), were transplanted into lethally-

irradiated (9.5Gy) recipients (Ly5.1).

CE-TOFMS analysis

For LSK cell analysis, non-transplanted LSK cells (Lineage marker-negative, Sca-1-positive and c-Kit-

positive) from BM pooled from three mice per group or donor-derived LSK cells of BM pooled from 25

mice per group werer sorted 16 hours after BMT, lysed to extract metabolites and subjected to mass

spectrometry analysis (Soga et al., 2006; Soga et al., 2003) .

For analysis of EML-C1 cells, cells were cultured with SB203580 or vehicle for 16 hours, lysed to extract

metabolites, and subjected to mass spectrometry analysis.

Statistical analysis

Data are presented as means ± SD, unless otherwise stated. Statistical significance was determined by

Tukey’s multiple comparison test. The two-tailed Student’s t-test was used for experiments with two

groups, the log-rank test was used to analyze survival data and chi-square test was used for frequency

distribution. The Smirnov-Grubb’s test was used to assess outliers.

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