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1 doi:.38/nture99 Dy Dy Dy 3 Dy 4 Bleed INDO INDO INDO INDO INDO INDO INDO INDO 3 7 CFU-GM/ml lood Indomethcin Biclein + Indo + Bcln c WBC PMN LYMPH MONO RBC PLT Ctrl 7.4±.9.4±.8.4±.39.37± ±. 76±3 Indo 6.63±.67.± ±..6±.9 8.4±.7 ±7 Supplementry Figure. Moiliztion regimens nd CBC fter indomethcin tretment., Schemtic of the dosing regimen used to evlute the moiliztion of CFU-GM using, Indomethcin, or the comintion of plus Indomethcin., Moiliztion of hemtopoietic progenitors y Indomethcin,, or comintion tretment (n= mice per group, ech ssyed individully). c, Complete lood counts in control or AID treted mice. P<.; unpired two-tiled t-test.
2 doi:.38/nture99 SC-6 Vleryl Slicylte Indomethcin Fold Chnge over mg/kg, id Fold Chnge over mg/kg, id Fold Chnge over..... mg/kg, id Aspirin Iuprofen Fold Chnge over mg/kg, id Fold Chnge over mg/kg, id Celecoxi Vldecoxi Fold Chnge over mg/kg Fold Chnge over mg/kg, id Fold Chnge over... mg/kg, id Supplementry Figure. Enhncement in HPC niche egress requires inhiition of oth COX- nd COX-. Mice were moilized y 4 dy regimen of ( µg/kg, twice dily) with co-dministrtion of AIDs rnging in COX nd COX selectivity. Dt re expressed s fold chnge over G- CSF lone, n=4 or mice per group, ech ssyed individully. P<., P<.; one-wy ANOVA with Bonferroni post-hoc nlysis. All error rs represent men ± s.e.m.
3 doi:.38/nture99 Eicosnoid (ng) per femur PGF 6-KPGF PGE TxB PGD 3% Inhiition 7% Inhiition Reltive COX Activity COX COX Supplementry Figure 3. inhiits oth COX- nd COX- in the one mrrow of mice., Anlysis of COX- nd COX- derived eicosnoids in the one mrrow of mice fter tretment with meloxicm or vehicle control., Functionl ssessment of COX- nd COX- ctivity from one mrrow cells fter tretment with meloxicm or vehicle control. n= mice per group, ech ssyed individully. P<., P<., P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m. 3
4 doi:.38/nture99 c Fold Increse in CFU-GM/mL Blood over CFC / Spleen x Indomethcin CFU-GM BFU-E CFU-GEMM CFC d Asolute Cell Numer / ml Blood 3 Asolute Numer / Spleen SKL x 4 + Indomethcin SKL / ml Blood x SLAM SKL / ml Blood SLAM SKL x 3 Control + Supplementry Figure 4. Indomethcin nd meloxicm increse niche egress of oth functionlly nd phenotypiclly defined HPC nd HSC., Moiliztion of CFU-GM with, or the comintion of nd Indomethcin. Dt re expressed s men ± s.e.m. from 3 experiments, n= mice totl per group, ech ssyed individully., Flow cytometric nlysis of phenotypiclly defined HSC in peripherl lood of mice treted with or the comintion of nd Indomethcin. (n= mice per group, ech ssyed individully) c, Moiliztion of CFC with meloxicm, or the comintion of +. (n=4- mice per group, per experiment; 3 seprte experiments). d, Moiliztion of phenotypiclly defined HSC in peripherl lood of mice treted with or the comintion of nd. (n=4- mice per group, per experiment; 3 seprte experiments). Dt re expressed s men ± s.e.m P<., P<., P<. unpired two tiled t-test. 4
5 doi:.38/nture CFC/ml Blood x 4 6 SLAM-SKL/ml Blood x Wild Type -ALOX -/- Wild Type -ALOX -/- + (-ALOX -/- ) + (-ALOX -/- ) Supplementry Figure. Enhnced HSC nd HPC moiliztion in -ALOX knockout mice., Moiliztion of CFC nd, SLAM SKL cells with or + meloxicm in wild type or - ALOX knockout mice. n=4 mice per group, ech ssyed individully. P<., P<.; one-wy ANOVA with Bonferroni post-hoc nlysis. All error rs represent men ± s.e.m.
6 doi:.38/nture99 Dy Dy Dy 3 Dy 4 Bleed AMD CFC / ml Blood CFU-GM CFU-GEMM BFU-E MXC MXC MXC MXC MXC MXC MXC MXC AMD vehicle AMD3 AMD+ M + M AMD+ AMD + + M c d KL cells/ml lood x 3 SKL cells/ml lood x : 4 3 : AMD3 + AMD AMD3 + AMD e f CLP/ml lood x 4 3 : CMP/ml lood x : AMD3 + AMD AMD3 + AMD Supplementry Figure 6. Enhnced HSC nd HPC moiliztion with AMD3., Schemtic of the dosing regimens used to evlute AID enhncement of AMD3 moiliztion. Mice were treted with meloxicm, AMD3, AMD3 + meloxicm,, G- CSF + meloxicm, AMD3 +, or AMD3 + + meloxicm nd peripherl lood, colony forming cells, c, c-kit + linege - (KL) cells, d, Sc- + c-kit + linege - (SKL) cells, e, c-kit low Sc- low IL-7Rα + Flk hi linege -, common lymphoid progenitors (CLP), nd f, c-kit + Sc- - CD34 + FcγR low linege -, common myeloid progenitors (CMP), were determined (n= mice per group, ech ssyed individully). P<., P<., P<., one-wy ANOVA, Tukey-Krmer post-hoc nlysis. All error rs represent men ± s.e.m. 6
7 doi:.38/nture99 c Tret CD4. mice with or + CD4. Competitors Collect peripherl lood mononucler cells Hrvest one mrrow from CD4. mice Dy Dy Dy 3 Dy 4 Dy Dy 6 Trnsplnt x 6 PBMC competitively ginst x WBM into lethlly irrdited recipients Bleed CRU per x 6 PBMC. + AID..... MXC MXC MXC MXC No Stgger MXC MXC MXC MXC Dy Stgger MXC MXC MXC MXC Dy Stgger d % of Control MFI of CXCR4 on SKL Cells Control + AID (No Stgger) + AID ( Dy Stgger) + AID ( Dy Stgger) e Asolute Numer / ml Blood 3 3 SKL x SLAM SKL + Supplementry Figure 7. Stggered dosing regimens restore peripherl HSC/HPC CXCR4 receptor expression., BoyJ mice were treted with with or without AID for 4 dys. On dy, LDMC from peripherl lood were cquired nd trnsplnted t : rtio with C7Bl/6J WBM competitors into lethlly irrdited C7Bl/6J recipient mice., Competitive repopulting units were clculted 3 months post-trnsplnt (men ± s.e.m.). Dt represent pooled experiments, n= mice per group, per experiment, ech ssyed individully. c, Schemtic of stggered dosing regimens of with (MXC). A 4-dy regimen of ws either co-dministered with for 4 dys (no stgger) or ws stggered or dys to llow for restortion of endogenous PGE iosynthesis. d, Mice were led nd peripherl lood mononucler cells (PBMC) were stined for SKL nd the CXCR4 receptor nd evluted y FACS. Dt represent the percent of control men fluorescence intensity (MFI) for CXCR4 expression on SKL cells, expressed s men ± s.e.m., n= mice per group, ech ssyed individully. e, Assessment of SKL nd SLAM SKL cells in the lood fter or + meloxicm (-dy stgger) (n=4 mice per group, ech ssyed individully). P<., P<.; unpired two-tiled t-test. 7
8 doi:.38/nture99 week % Chimerism PBMC 3 3 G + M (No stgger) G + M ( dy stgger) G + M ( dy stgger) : : 3: 4: PBMC to Competitor BM Rtio CRU per x 6 PBMC 3 % Negtive Recipients 37 Numer of Moilized LDMN Trnsplnted.x 6 x 6.x 6 x 6.x 6 /7,736 P=.93 /48,63 P=.3 /,74 4 week % Chimerism PBMC 3 3 G + M (No stgger) G + M ( dy stgger) G + M ( dy stgger) : : 3: 4: PBMC to Competitor BM Rtio CRU per x 6 PBMC 3 % Negtive Recipients 37 Numer of Moilized LDMN Trnsplnted.x 6 x 6.x 6 x 6.x 6 /6,383 P=.8 /48,63 P=. /8,66 Supplementry Figure 8. Enhnced long-term competitive repopultion with AID moilized grfts. Chimerism, competitive repopulting units (CRU), nd long-term repopulting HSC frequency (Poisson distriution) nd 4 weeks fter limiting dilution competitive trnsplnts of PBMC from mice moilized with nd comintion regimens, n=8 mice per group, ech ssyed individully. P<.; unpired two-tiled t-test. 8
9 doi:.38/nture99 % Chimerism Weeks % WBC T Cells B Cells Myeloid Supplementry Figure 9. Enhnced secondry repopultion with AID moilized grfts Bone mrrow from mice originlly trnsplnted with PBMC moilized y or + (see Figure f) ws secondrily trnsplnted nd, chimerism nd, multilinege reconstitution were determined (n= mice per group, ech ssyed individully). 9
10 doi:.38/nture Control Totl CFC per Femur CFU-GM BFU-E GEMM CFC Gted on Lin neg Sc- Sc-+IL7R c-kit Gted on Lin neg c-kit CD FCRll/lll CD CD34 c SLAM SKL cells / femurs x 3 (Gte ) 4 3 d % Chimerism e Megkryocyte-Erythroid Progenitor Cells (MEP)/ femurs x 3 (Gte ) f i Common Myeloid Progenitor Cells (CMP)/ femurs x 3 (Gte 3) Grnulocyte-Mcrophge Progenitor Cells (GMP)/ femurs x 3 (Gte 4) g h j k CFU per CMP SCF + GM-CSF CFU per CMP M-CSF CFU per GMP SCF + GM-CSF CFU per GMP M-CSF
11 doi:.38/nture99 Supplementry Figure. Enhnced one mrrow myeloid progenitors with no ltertion of HSCs in AID treted mice., Expnsion of one mrrow HPC fter meloxicm tretment (n=4 mice/group/experiment; two seprte experiments.), Representtive flow cytometry gting of SLAM SKL cells (gte ), megkryocyte erythroid progenitor (MEP) cells (gte ), common myeloid progenitor (CMP) cells (gte 3) nd grnulocyte mcrophge progenitor (GMP) cells. c, SLAM SKL cell numer etween vehicle nd meloxicm treted mice (n=4 mice per group, ech ssyed individully). d, Chimerism 6 weeks post-trnsplnt fter competitive : trnsplnt of vehicle or stggered meloxicm ( dy stgger) treted mice (n= mice per group, ech ssyed individully. e, MEP numer in vehicle or meloxicm treted mice. (n=4 mice per group, ech ssyed individully). f, CMP numer in vehicle or AID treted mice (n=4 mice per group, ech ssyed individully). CMPs were sorted nd plted in g, GM-CSF ng/ml) + SCF ( ng/ml) or h, M-CSF ( ng/ml), nd CFU per cells determined (n= pltes per tretment nd condition group). i, GMP numer in vehicle or AID treted mice (n=4 mice per group, ech ssyed individully). GMPs were sorted nd plted in j, GM-CSF + SCF or j, M-CSF, nd CFU per cells determined (n= pltes per tretment nd condition group). P<., P<., P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m.
12 doi:.38/nture99 Inhiition of CFU-M Compound Agonist Specificity IC (nm) Rtio PGE EP, EP, EP3, EP4.37±.3 dmpge EP, EP3, EP4 3.± phenyl trinor PGE EP, EP3 39± Butprost EP 89,±4 37,9 Sulprostone EP3, EP 699± deoxy PGE EP, EP3, EP4.± 4.8 L-9,688 EP4.9±.7. PGE.4±.3 + µm AH3848 EP4 ntgonist.6± µm AH ± µM AH ± µM AH ± Schild Plot K.69x -6 M: R =.9 A/A =-.7: = Competitive Antgonist % CFU-M of Control Control EP -/- EP -/- EP3 -/- EP4 +/- (Het) EP4 -/- c % CFU-M of Control Concentrtion of PGE (M) d e f CFU-GM/ml Blood SKL/mL Blood x SLAM SKL/mL Blood EP4 fl/fl (WT) EP4 Cre fl/fl (KO) Supplementry Figure. Prostglndin E EP4 receptor ntgonism or knockout expnds one mrrow progenitors nd enhnces progenitor nd stem cell moiliztion, Inhiition of CFU-M formtion in the presence of PGE gonists nd ntgonists., Inhiition of CFU-M formtion y PGE from one mrrow of wild type, EP -/-, EP -/-, EP3 -/-, EP4 +/-, or EP4 -/- one mrrow cells. c, Incresed CFU-M in one mrrow of EP4 -/- mice. d, Conditionl deletion of EP4 results in enhnced CFU-GM, e, SKL cells nd f, SLAM SKL cells in the peripherl lood (n=3 mice per group, ech ssyed individully). P<., P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m.
13 doi:.38/nture c d Osteolinege cell width (microns) CFU-GM/femur x CFU-GM/ml lood C 3 4 # dys of AID dosing Y=-.367x+3. R=.87; P<. C 3 4 # dys of AID dosing Y=-9x+8.763x 3 R=.8; P<. C 3 4 # dys of AID dosing Y=9.3x-64 R=.8; P<. Supplementry Figure. Progressive ttenution of endostel lining osteolinege cells fter AID tretment., Attenuted endostel lining osteolinege cells fter -4 dys of AID tretment. Originl mgnifiction 4X., Osteolinege cell width fter -4 dys of AID tretment ( mesured osteolinege cells per mouse femur, n=4- mice per group, ech ssyed individully). c, Expnsion of one mrrow hemtopoietic progenitors, nd d, incresed progenitor moiliztion fter -4 dys. 3
14 doi:.38/nture99 Control AID c d e f Supplementry Figure 3. Enhnced myelopoiesis nd ttenuted osteolinege cells fter AID tretment. Mice were treted with 4 dy regimen of vehicle or AIDs, femurs were cquired, sectioned nd H&E stined. Shown is the mrrow from, control nd, AID treted mice, showing incresed myelopoiesis nd reduced erythropoiesis fter tretment. c, Control mice mintined norml osteolinege cell lining of one (rrows) in the diphysel region of the femur, while d, AID treted mice showed less distinct lyer of osteolinege cells, with mrked flttening. e, Control mice lso hd norml osteolinege lining within the epiphysel region of the femur, while f, AID treted mice showed less distinct lyer of osteolinege cells, with mrked flttening. 4X mgnifiction. 4
15 doi:.38/nture99 WT EP4 fl/fl EP4 Cre fl/fl Tmoxifen Treted.7 WT EP4 fl/fl EP4Cre fl/fl c 3 O/Bone Surfce #O/Bone Surfce Supplementry Figure 4: Endostel lining osteolinege cells in EP4 conditionl knockout mice., Shown re endostel lining regions from wild type mouse compred to n EP4 fl/fl or EP4 Cre fl/fl (knockout) 4 dys fter tmoxifen tretment., Conditionl deletion of EP4 resulted in reduced proportion of osteolinege lining one surfces nd c, osteolinege numer. (n=3 mice per group, ech ssyed individully). P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m.
16 doi:.38/nture99 Control c g h i Control d e f MS/BS% OS/BS% MAR OS(OLC+)/BS% j k l BFR QS/BS%. Supplementry Figure. Reduced osteolinege function in AID treted ones. Clcein uptke during dynmic one formtion ssy in, control,, meloxicm, or c, treted mice, respectively, demonstrtes reduced d, minerlized surfces, e, minerl pposition rte (MAR) nd f, one formtion rte (BFR) fter tretment with meloxicm or (n= mice per group). g, h, i, Modified Goldner's trichrome stining for osteoid one in control, meloxicm or G- CSF treted mice, respectively, demonstrtes reduced j, osteoid surfce (OS), k, osteoid/osteolinege cell (OLC) positive surfces, nd l, incresed quiescent (QS) one surfces fter tretment with or (n= mice per group, ech ssyed individully). P<., unpired two-tiled t-test. P<. compred to. All error rs represent men ± s.e.m. 6
17 doi:.38/nture99 c #OC / BPm, mm Control + Control + ZA + ZA + ZA + + ZA d Colonies per ml Blood x 3 Asolute Numer / ml Blood ZA: CFU-GM BFU-E CFU-GEMM CFC ZA: SKL x SLAM SKL e 4 3 Colonies per Spleen x Asolute Numer / Spleen 4 3 ZA: CFU-GM BFU-E CFU-GEMM CFC ZA: SKL x 4 SLAM SKL x 3 Supplementry Figure 6. Osteoclst function does not lter meloxicm enhncement of moiliztion., Osteoclst (OC) numer fter tretment with vehicle, meloxicm, or + meloxicm (n= mice per group, ech ssyed individully)., Assessment of HPC in the peripherl lood nd c, spleen, compring moiliztion strtegies with or without zoledronic cid tretment. d, Assessment of SKL nd SLAM SKL cells in the peripherl lood nd e, spleen, compring moiliztion strtegies with or without zoledronic cid tretment (n= mice per group, ssyed individully.) ). P<., P<., P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m. 7
18 doi:.38/nture99 Control + c Control Gr- F4/8 Gr- - F4/8 + M-CSFR low cells/ femur( 4 ) M-CSFR M-CSFR Supplementry Figure 7. No ltertion in one mrrow-resident mcrophges in AID treted mice., Immunohistochemicl stining for resident F4/8 + one mrrow mcrophges. Originl mgnifiction X., Representtive flow gting of one mrrow mcrophges. c, Reduced mcrophges fter, ut not meloxicm tretment s determined y FACS (n= mice per group, ech ssyed individully). P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m. 8
19 doi:.38/nture99 Gted on EC VEGFR3 FSC FSC VEGFR3 FSC FSC CD4 Ter9 Sc- CD3 Live-ded dye/ Annexin V + EC per femur 4 6 % Live/ded/Annexin V + SE Supplementry Figure 8. No ltertions in sinusoidl endothelil cell numer or poptosis in AID treted one mrrow. Shown re representtive flow cytometry plots of vehicle or AID treted mice for nlysis of one mrrow sinusoidl endothelil cell numer nd poptosis (n= mice/group, ssyed individully). All error rs represent men ± s.e.m. 9
20 doi:.38/nture99 VEGFR3 Stining Exmple Sinusoid Volume Vessel Volume / Tissue Volume % Vessel Numer / Tissue Volume mm 3 3 Endothelil Numer / Tissue Volume mm Supplementry Figure 9. No ltertions in sinusoid vessel numer, volume or endothelil cell numer in AID treted one mrrow. Shown is representtive IHC nlysis of VEGFR3 stined sinusoids, showing n exmple sinusoid volume (red outline), with nlysis of vessel volume, vessel numer nd endothelil cell numer etween meloxicm nd vehicle treted mice. (n=3,4 mice per group, ech ssyed individully). All error rs represent men ± s.e.m.
21 doi:.38/nture99 Nestin-GFP+ Cells/ Femurs + Tiis Supplementry Figure. No ltertion in Nestin+ cell numer in AID treted one mrrow. Nestin-GFP mice were treted with vehicle or meloxicm for 4 dys nd GFP + cells ssessed y flow cytometry (n=3 mice per group, ech ssyed individully). All error rs represent men ± s.e.m.
22 doi:.38/nture99 CD66 c CD4 - /TER9 - /CD3 - Sc- Lin- CD66+ Sc-- Reltive Expression (Log Scle) EP Receptor EP Receptor EP3 Receptor EP4 Receptor.... CD66+ Sc-- CD66- Sc-+ CD66- Sc-- Lin- CD66- Sc-+ Lin- CD66- Sc-- %mrna Jgd Rnx VCM SCF SDF OPN ND ND ND Jgd Rnx VCM SCF SDF OPN Jgd Rnx VCM SCF SDF OPN d Sc-+ Alcm- cells/ femur x 3 WT EP4 fl/fl EP4cre fl/fl Supplementry Figure. AIDS reduce osteolinege cell production of hemtopoietic supportive molecules nd mesenchyml progenitor cells re reduced in EP4 knockout mice., Representtive gting strtegy for osteolinege cell popultions. "Linege (Lin)" refers to CD4, CD3 nd TER9., Reltive EP receptor expression on osteolinege cells (n= mice per group, ech ssyed individully). c, Expression nd regultion of HSC mintennce genes y osteolinege cells; Jgged- (Jgd), Runx- (Rnx), VCAM- (VCM), stem cell fctor/kit lignd (SCF), stroml derived fctor- (SDF), nd osteopontin (OPN). n=4 independent groups per condition. d, Anlysis of CD4/TER9/CD3 - Sc- + Alcm - cells in WT, or tmoxifen treted EP4 fl/fl nd EP4 Cre fl/fl (knockout mice) (n=4 mice per group, ech ssyed individully). P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m.
23 doi:.38/nture99 c Colonies per ml Blood Asolute Numer / ml Blood WT KO WT KO WT KO WT KO CFU-GM BFU-E CFU-GEMM CFC WT KO WT KO SKL x SLAM SKL d Cells per Spleen Colonies per Spleen WT KO WT KO WT KO WT KO CFU-GM BFU-E CFU-GEMM CFC SKL x SLAM SKL Control + Control CXCR4 -/- CXCR4 /- CXCR4 -/- + CXCR4 -/- Supplementry Figure. HPC nd cndidte HSC moiliztion in CXCR4 nd knockout mice. CFU-GM, BFU-E, nd CFU-GEMM moiliztion in CXCR4 knockout mice or Cre(-) littermte controls in, peripherl lood or, spleen, nd HPC nd HSC moiliztion to c, peripherl lood or d, spleen (n= mice per group, ech ssyed individully). P<., P<., P<.; unpired two-tiled t- test. All error rs represent men ± s.e.m. 3
24 doi:.38/nture99 Colonies per Femur Colonies per ml Blood WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO CFU-GM BFU-E CFU-GEMM CFC CFU-GM BFU-E CFU-GEMM CFC Control c 3 d Asolute Numer / ml Blood Control OPN -/- OPN -/- OPN -/- + OPN -/- WT KO WT KO SKL x SLAM SKL e Cells per Spleen Colonies per Spleen 3 WT KO WT KO WT KO WT KO CFU-GM BFU-E CFU-GEMM CFC SKL x SLAM SKL Supplementry Figure 3. HPC nd cndidte HSC moiliztion in OPN knockout mice., Bone mrrow, peripherl lood nd c, spleen CFU-GM, BFU-E, nd CFU-GEMM in wild type or OPN knockout mice efore nd fter tretment nd HSC moiliztion to d, peripherl lood nd e, spleen in OPN knockout mice fter tretment (n=4 mice per group, ech ssyed individully). P<., P<., P<.; unpired two-tiled t-test. All error rs represent men ± s.e.m. 4
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