1. Stem Cells and Regenerative Medicine
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Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

  1. Bo Shen
  2. Kristy Vardy
  3. Payton Hughes
  4. Alpaslan Tasdogan
  5. Zhiyu Zhao
  6. Rui Yue
  7. Genevieve M Crane
  8. Sean J Morrison  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Tongji University, China
  3. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Research Article
Cite this article as: eLife 2019;8:e42274 doi: 10.7554/eLife.42274
7 figures, 2 tables and 2 additional files

Figures

Osteolectin contains conserved integrin binding motifs and binds with high affinity to integrin α11β1.

(A, B) The human (A) and mouse (B) Osteolectin proteins contain RGD and LDT sequences. (C) Alignment of Osteolectin amino acid sequences shows that the RGD and LDT domains are evolutionarily conserved among bony vertebrates. (D, E) RNA-seq analysis of integrin α (D) and β (E) subunits in PDGFRα+CD45-Ter119-CD31- bone marrow stromal cells from enzymatically dissociated adult bone marrow (n = 2 independent samples). These cells are uniformly positive for LepR expression (Zhou et al., 2014). (F) RNA-seq analysis of Itga1, Itga6, Itga11, and Itgav in PDGFRα+CD45-Ter119-CD31- bone marrow stromal cells, VE-Cadherin+ bone marrow endothelial cells, and whole bone marrow cells (n = 2 independent samples per cell population). (G) Itga11 expression in cell populations from mouse bone marrow by qRT-PCR (n = 3 independent samples per cell population). The markers used for the isolation of each cell population are shown in Supplementary file 1. (H) In MC3T3-E1 preosteoblast cells expressing Flag-tagged Osteolectin, anti-Flag antibody co-immunoprecipitated endogenous integrin β1 and integrin α11 with Flag-tagged Osteolectin (results are representative of two independent experiments). (I) Recombinant human Osteolectin (rhOln) selectively bound to recombinant human integrin α11β1 and α10β1, but not to other integrins (n = 3 independent experiments). (J) Integrin α11β1 bound Osteolectin and recombinant human Pro-Collagen 1α (rhCol1A) with similar affinities, but not bovine serum albumin (BSA) (n = 3 independent experiments). (K) Osteolectin, but not Pro-Collagen 1α, promoted osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells (n = 3 independent experiments). (L) 200 nM RGDS peptide inhibited the binding of integrin α11β1 to recombinant human Osteolectin. (M) 100 μM RGDS peptide inhibited osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells in response to 30 ng/ml of recombinant human Osteolectin. All numerical data reflect mean ±standard deviation. Statistical significance was determined with one-way (G) or two-way ANOVAs with Dunnett’s multiple comparisons tests (K) or Tukey’s multiple comparisons tests (M).

https://doi.org/10.7554/eLife.42274.003
Osteolectin activates Wnt pathway signaling in skeletal stem/progenitor cells.

(A) Western blot of cell culture supernatant from parental or Osteolectin deficient MC3T3-E1 cells and human bone marrow stromal cells (hBMSC#1 and hBMSC#2 cells) (this blot is representative of blots from three independent experiments). (B) Osteogenic differentiation in culture of parental or Osteolectin deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells. Alizarin red staining was performed after 14 days (for MC3T3-E1 cells) or 21 days (for hBMSC cells) to quantify osteoblast differentiation and mineralization (n = 3 independent experiments). (C) qRT-PCR analysis of Dmp1 transcript levels in MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells after 14 to 21 days of osteogenic differentiation (n = 5 independent experiments). (D) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were stimulated with recombinant mouse Osteolectin (rOln) in osteogenic differentiation medium then lysed 0, 15, 30, or 60 min later and immunoblotted for phospho-PI3K, phospho-Akt, phospho-GSK3, β-catenin, total GSK3, and Actin (results are representative of 3 independent experiments). (E) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium for 24 hr with PBS or recombinant mouse Osteolectin, lysed, and immunoblotted for phospho-GSK3, β-catenin, and Actin (this blot is representative of blots from three independent experiments). (F) qRT-PCR analysis of Wnt target gene transcript levels (Alkaline phosphatase, Axin2, Lef1, or Runx2) in MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells 24 hr after transfer into osteogenic differentiation medium, with PBS or recombinant mouse Osteolectin (n = 5 independent experiments). (G) Parental or Osteolectin deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells after 24 hr in osteogenic differentiation medium were lysed and immunoblotted for phospho-GSK3, β-catenin, and Actin (this blot is representative of blots from three independent experiments). (H) qRT-PCR analysis of Wnt target gene transcript levels in parental or Osteolectin deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells 24 hr after transfer into osteogenic differentiation medium (n = 5 independent experiments). All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with two-way ANOVAs with Sidak’s multiple comparisons tests.

https://doi.org/10.7554/eLife.42274.005
Osteogenic differentiation in response to Osteolectin requires β-catenin.

(A and B) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml recombinant mouse Osteolectin, as well as DMSO or 200 nM of the GSK3 inhibitor AZD2858. (A) Cells were lysed 24 hr later and immunoblotted for phospho-GSK3, β-catenin, and Actin. (B) Alizarin red staining after 14 days (MC3T3-E1 cells) or 21 days (hBMSC cells) to quantify osteoblast differentiation and mineralization (n = 3 independent experiments). (C and D) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml Osteolectin, as well as DMSO or 200 nM of the β-catenin inhibitor IWR-1-endo. (C) Cells were lysed 24 hr later and immunoblotted for phospho-GSK3, β-catenin, and Actin. (D) Alizarin red staining after 14 days (MC3T3-E1 cells) or 21 days (hBMSC cells) to quantify osteoblast differentiation and mineralization (n = 3 independent experiments). All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with two-way ANOVAs with Tukey’s multiple comparisons tests.

https://doi.org/10.7554/eLife.42274.007
Integrin α11 is required for osteogenic differentiation and Wnt pathway activation in response to Osteolectin but exogenous collagen does not activate Wnt pathway signaling.

(A) Osteogenic differentiation in culture of parental or Itga11 deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells with PBS or recombinant mouse Osteolectin (n = 3 independent experiments). (B) qRT-PCR analysis of Dmp1 transcript levels in MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells after 14 to 21 days of osteogenic differentiation (n = 5 independent experiments). (C) Parental or Itga11 deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with or without recombinant mouse Osteolectin then lysed 24 hr later and immunoblotted for phospho-GSK3, β-catenin, and Actin (this blot is representative of blots from three independent experiments). (D) qRT-PCR analysis of Wnt target gene transcript levels in parental or Itga11 deficient MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells 24 hr after transfer into osteogenic differentiation medium with PBS or Osteolectin (n = 5 independent experiments). (E) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml Osteolectin, or 30 ng/ml recombinant Pro-Collagen 1α, then lysed 24 hr later and immunoblotted for phospho-GSK3, β-catenin, and Actin (this blot is representative of blots from three independent experiments). (F) qRT-PCR analysis of Wnt target gene transcript levels in MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells 24 hr after transfer into osteogenic differentiation medium with PBS or 30 ng/ml Osteolectin, or 30 ng/ml Pro-Collagen 1α (n = 5 independent experiments). (G) Primary mouse bone marrow stromal cells were adherently cultured in osteogenic differentiation medium. PBS (control), recombinant human Pro-Collagen 1α, or recombinant human Osteolectin was added, then the cells were lysed 1 hr later and lysates were immunoblotted for phospho-FAK, phospho-GSK3, total FAK, and Actin (this blot is representative of blots from three independent experiments). (H) Primary mouse bone marrow stromal cells adherently cultured in osteogenic differentiation medium were treated with PBS (control), recombinant human Pro-Collagen 1α, or recombinant human Osteolectin then lysed 24 hr later and lysates were immunoblotted for phospho-FAK, phospho-GSK3, β-catenin, total FAK, and Actin (this blot is representative of blots from three independent experiments). (I) Primary mouse bone marrow stromal cells adherently cultured in osteogenic differentiation medium were treated with 30 ng/ml recombinant human Osteolectin or PBS (control) then lysed 2, 4, 6, 12, or 24 hr later. Nuclear and cytosolic/membrane-associated fractions were isolated from lysates by centrifugation then immunoblotted for β-catenin. As loading controls, Histone H3 was blotted in the nuclear fraction and Actin was blotted in the cytosolic/membrane-associated fraction (this blot is representative of blots from three independent experiments). (J) qRT-PCR analysis of Ctnnb1 transcript levels in cells from the experiment in panel (I) (n = 3 independent experiments). (K) Primary mouse bone marrow stromal cells from Lepr-Cre; Itga11fl/fl or littermate control mice were adherently cultured in osteogenic differentiation medium. PBS (control), recombinant human Pro-Collagen 1α, or recombinant human Osteolectin was added then the cells were lysed 1 hr later and lysates were immunoblotted for phospho-FAK and FAK (this blot is representative of blots from two independent experiments). All numerical data reflect mean ±standard deviation. Statistical significance was determined with two-way ANOVAs with Tukey’s multiple comparisons tests (A, B and D), Dunnett’s multiple comparisons tests (F), or Sidak’s multiple comparisons tests (J).

https://doi.org/10.7554/eLife.42274.009
Figure 5 with 1 supplement
Conditional Itga11 deletion from LepR+cells accelerates the loss of trabecular bone during aging.

(A) Lepr-Cre; Itga11fl/fl mice were grossly normal and indistinguishable from littermate controls. (B – D) Body length (B), body mass (C) and femur length (D) did not significantly differ between Lepr-Cre; Itga11fl/fl mice and sex-matched littermate controls at 2, 6, or 12 months of age (n = 4–7 mice per genotype per sex per time point, from at least three independent experiments). (E) ELISA measurement of serum Osteolectin levels in Lepr-Cre; Itga11fl/fl mice and littermate controls at 2, 6, and 12 months of age (n = 8 mice per genotype per time point from four independent experiments). (F) Representative microCT images of trabecular bone in the distal femur metaphysis of male Lepr-Cre; Itga11fl/fl mice and littermate controls at 2, 6, and 12 months of age. (G–L) microCT analysis of trabecular bone volume/total volume (G), trabecular number (H), trabecular bone thickness (I), trabecular bone spacing (J), connectivity density (K), and bone mineral density (L) in the distal femur metaphysis of Lepr-Cre; Itga11fl/fl mice and sex-matched littermate controls at 2, 6, and 12 months of age (n = 4–7 mice per genotype per sex per time point from at least three independent experiments). (M) Trabecular bone mineral apposition rate based on calcein double labelling in the distal femur metaphysis (n = 3 mice per genotype per sex per time point). (N) Bone resorption rate analysis by measuring the deoxypyridinoline/creatinine ratio in the urine of LepR-Cre; Itga11fl/fl mice and littermate controls at 6 and 12 months of age (n = 4–5 mice per genotype per time point from three independent experiments). (O) Serum P1NP levels in LepR-Cre; Itga11fl/fl mice and sex-matched littermate controls at 6 and 12 months of age (n = 4 mice per genotype per time point from three independent experiments). All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with two-way ANOVAs with Sidak’s multiple comparisons tests.

https://doi.org/10.7554/eLife.42274.011
Figure 5—figure supplement 1
Generation of an Itga11 floxed mice.

(A) DNA surrounding Itga11 exon3 was used as homologous arms for recombineering. The recombineering plasmid was modified by inserting loxP sites flanking exon3. Deletion of exon three leads to a frame shift that would be expected to give a complete loss of α11 function (Popova et al., 2007). The loxP insertion sites were chosen so as not to disrupt sequences conserved among species. Cas9 mRNA, sgRNAs, and the recombineering plasmid were microinjected into C57BL/Ka zygotes. Correctly-targeted founder mice (F0) were identified by Southern blotting (B) using an internal (3’) probe and an external (5’) probe to ensure the recombineering vector was integrated in the correct genomic location, and not in other regions of the genome. Mice were backcrossed at least three times onto a C57BL/Ka background before analysis. (C) PCR genotyping demonstrated germline transmission of the Itga11flox allele. (D) qRT-PCR analysis of Itga11 transcript levels in LepR+ cells from the bone marrow of Lepr-Cre; Itga11fl/fl and sex-matched littermate controls (n = 4 mice per genotype per time point from two independent experiments). All numerical data reflect mean ±standard deviation.

https://doi.org/10.7554/eLife.42274.013
Conditional Itga11 deletion from LepR+cells reduces cortical bone formation in adult mice.

(A) Representative microCT images of cortical bone in the mid-femur diaphysis of male Lepr-Cre; Itga11fl/fl mice and littermate controls at 2, 6, and 12 months of age. (B–F) microCT analysis of the total area (B), cortical area (C), cortical thickness (D), cortical area/total area (E), and cortical bone mineral density (F) in the mid-femur diaphysis of Lepr-Cre; Itga11fl/fl mice and sex-matched littermate controls at 2, 6, and 12 months of age (n = 4–7 mice per genotype per sex per time point from at least three independent experiments). (G) Cortical bone mineral apposition rate based on calcein double labelling in the mid-femur diaphysis (n = 3–4 mice per genotype per sex per time point from three independent experiments). All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with two-way ANOVAs with Sidak’s multiple comparisons tests.

https://doi.org/10.7554/eLife.42274.015
Figure 7 with 1 supplement
Integrin α11 is required by bone marrow stromal cells to undergo osteogenesis in response to Osteolectin.

(A) CFU-F frequency and (B) cells per CFU-F colony formed by bone marrow cells from Lepr-Cre; Itga11fl/fl mice and littermate controls at 2 and 6 months of age (n = 6–8 mice per genotype per time point from at least three independent experiments). (C–E) Osteogenic (C); n = 7 mice per genotype, total, from seven independent experiments), adipogenic (D); n = 6 mice per genotype, total, from six independent experiments), and chondrogenic (E); n = 5 mice per genotype, total, from five independent experiments) differentiation of bone marrow stromal cells cultured from the femurs of Lepr-Cre; Itga11fl/fl mice and sex-matched littermate controls at 2 months of age. (F) Recombinant mouse Osteolectin promoted osteogenic differentiation in culture by femur bone marrow stromal cells from control but not Lepr-Cre; Itga11fl/fl mice (n = 3 mice per genotype, total, from three independent experiments). (G) qRT-PCR analysis of Dmp1 transcript levels in cells from panel (F). (H–N) Subcutaneous injection of recombinant mouse Osteolectin daily for 28 days (50 μg/kg body mass/day) significantly increased trabecular bone volume and number in female control mice but not female Lepr-Cre; Itga11fl/fl mice. (H) Representative microCT images of trabecular bone in the distal femur metaphysis. (I–N) microCT analysis of trabecular bone volume/total volume (I), trabecular number (J), trabecular thickness (K), trabecular spacing (L), connectivity density (M), and bone mineral density (N) in the distal femur metaphysis (n = 5 mice per treatment, total, from three independent experiments). (O) The GSK3 inhibitor, AZD2858, rescued the osteogenic differentiation in culture of bone marrow stromal cells from the femurs of Lepr-Cre; Itga11fl/fl mice (n = 6 independent experiments in which cells from one mouse of each genotype were cultured in each experiment). (P) Western blotting of cultured cell lysates showed that AZD2858 promoted GSK3 phosphorylation and increased β-catenin levels in bone marrow stromal cells from Lepr-Cre; Itga11fl/fl mice and littermate controls. All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with Wilcoxon’s test followed by Holm-Sidak multiple comparisons adjustment (A and B), one-way (F), (G,) and O) ANOVAs with Sidak’s multiple comparisons tests, by paired t-tests (C–E), or by one-way ANOVAs with Tukey’s multiple comparisons tests (I–N).

https://doi.org/10.7554/eLife.42274.017
Figure 7—figure supplement 1
Osteolectin promotes Wnt target gene transcription in mouse bone marrow stromal cells in an integrin α11-dependent manner.

(A) Bone marrow stromal cells from Lepr-Cre; Itga11fl/fl mice and sex-matched littermates at 2 months of age were cultured in osteogenic differentiation medium supplemented with PBS (control) or 30 ng/ml Osteolectin and qRT-PCR analysis of Wnt target gene transcript levels was performed 24 hr later (n = 3 independent experiments). (B) Recombinant mouse Osteolectin subcutaneously injected daily for 28 days (50 μg/kg body mass/day) increased Wnt target gene transcript levels in LepR+ bone marrow cells from control, but not Lepr-Cre; Itga11fl/fl, mice (n = 4 mice per genotype per time point from two independent experiments). All numerical data reflect mean ±standard deviation. Statistical significance was assessed with two-way ANOVAs with Tukey’s multiple comparisons tests (A) or Dunnett’s multiple comparisons tests (B).

https://doi.org/10.7554/eLife.42274.019

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent(M.musculus)Osteolectin-/-PMID: 27976999
Genetic reagent (M.musculus)Lepr-CrePMID: 11283374JAX Stock (008320)
Antibodyrabbit polyclonal anti-phospho-PI3 KinaseCell Signaling4228S(1:1000)
Antibodyrabbit polyclonal anti-phospho-Akt (Ser473)Cell Signaling4060S(1:1000)
Antibodyrabbit polyclonal anti-phospho-GSK-3α/βCell Signaling9331S(1:1000)
Antibodyrabbit polyclonal anti-β-CateninCell Signaling8480S(1:1000)
Antibodyrabbit polyclonal anti-anti-β-ActinCell Signaling8457S(1:10000)
Antibodyrabbit polyclonal anti-GSK-3bCell Signaling9315S(1:1000)
Antibodygoat polyclonal anti-rabbit IgG, HRP-linked antibodyCell Signaling7074S(1:5000)
Antibodyhorse polyclonal anti-mouse IgG, HRP-linked antibodyCell Signaling7076S(1:5000)
Antibodydonkey polyclonal anti-sheep IgG, HRP-linked antibodyR and D SystemsHAF016(1:5000)
Antibodyrabbit polyclonal anti-phospho-FAK (Y397)Cell Signaling3283S(1:1000)
Antibodyrabbit polyclonal anti-Histone H3Cell Signaling4499S(1:5000)
Antibodygoat polyclonal anti-mouse OsteolectinR and D SystemsAF3729(1:1000)
Antibodysheep polyclonal anti-human OsteolectinR and D SystemsAF1904(1:1000)
Antibodyrabbit polyclonal anti-AdiponectinAbcamab181699(1:1000)
Antibodyrabbit polyclonal anti-Integrin α11Abcamab198826(1:1000)
Antibodyrabbit polyclonal anti-FAKCell Signaling3285P(1:1000)
Antibodymouse monoclonal anti-6x-His Tag antibodyThermo Fisher ScientificMA1-135(1:1000)
Antibodyrat polyclonal anti-CD45-APCTonbo20–0451(1:200)
Antibodyrat polyclonal anti-Ter119-APCTonbo20–5921(1:200)
Antibodyrat polyclonal anti-CD31-APCBiolegend102410(1:200)
Antibodygoat polyclonal anti-Mouse Leptin R, BiotinR and D SystemsAF497(1:200)
Antibodymouse monoclonal anti-Streptavidin PEBiolegend410504(1:500)
Peptide, recombinant proteinBovine serum albuminSigma-AldrichA3156
Peptide, recombinant proteinrecombinant human pro-Collagen I α1R and D Systems6220 CL
Peptide, recombinant proteinrecombinant human osteolectinPMID: 27976999
Peptide, recombinant proteinrecombinant mouse osteolectinPMID: 27976999
Peptide, recombinant proteinrecombiant Integrin α11β1 proteinR and D Systems6357-AB
Peptide, recombinant proteinrecombiant Integrin α10β1 proteinR and D Systems5895-AB
Peptide, recombinant proteinrecombiant Integrin αVβ3 proteinR and D Systems3050-AV
Peptide, recombinant proteinrecombiant Integrin αVβ1 ProteinR and D Systems6579-AV
Peptide, recombinant proteinrecombiant Integrin α4β1 proteinR and D Systems5668-A4
Peptide, recombinant proteinrecombiant Integrin α9β1 proteinR and D Systems5438-A9
Peptide, recombinant proteinrecombiant Integrin αIIbβ3 ProteinR and D Systems7148-A2
Peptide, recombinant proteinrecombiant Integrin αMβ2 ProteinR and D Systems4047-AM
Chemical compound,drugDAPILife TechnologiesD1306
Chemical compound, drugTRIzol LS ReagentInvitrogen10296028
Chemical cmpound,drugCollagenase, Type 1WorthingtonLS004196
Chemical compound, drugDispase IIRoche DiagnosticD4693
Chemical compound, drugDNase ISigma-Aldrich10 104 159 001
Chemical compound, drugIWR-1-endoSigma-Aldrich681669
Chemical compound, drugAZD2858SelleckS7253
Chemical compound, drugY-27632 Rock inhibitorSelleckS1049
Chemical compound, drug4% paraformaldehyde in PBSThermo Fisher ScientificJ19943-K2
Cell lines (M.musculus)MC3T3-E1, subclone 4ATCCCRL-2593
Cell lines (H.sapiens)hBMSC#1ATCCPCS-500–012
Cell lines (H.sapiens)hBMSC#2LonzaPT-2501
Commercial assay or kitCell Culture Contamination Detection KitMolecular ProbesC-7028
Commercial assay or kitTMB stop solutionKPL95059–198
Commercial assay or kitSureBlue TMB Microwell Peroxidase SubstrateKPL95059–282
Commercial assay or kitHBSS, without Calcium and MagnesiumCorning21022CV
Commercial assay or kitHBSS, with Calcium and MagnesiumCorning21023CV
Commercial assay or kitCleanCap Cas9 mRNATriLinkL-7206
Commercial assay or kitMEGAshortscript T7 Transcription KitAmbionAM1354
Commercial assay or kitMEGAclear Transcription Clean-Up KitAmbionAM1908
Commercial assay or kitEDTA-free Protease Inhibitor CocktailSigma-Aldrich11 836 170 001
Commercial assay or kitECL Western Blotting SubstratePierce32106
Commercial assay or kitSuperScript III Reverse TranscriptaseInvitrogen18080044
Commercial assay or kitStemPro Osteogenesis Differentiation KitGibcoA1007201
Commercial assay or kitStemPro Adipogenesis Differentiation KitGibcoA1007001
Commercial assay or kitStemPro Chondrogenesis Differentiation KitGibcoA1007101
Commercial assay or kitMicroVue DPD ELISA KitQuidel8007
Commercial assay or kitMicroVue Creatinine Assay KitQuidel8009
Commercial assay or kitRat/Mouse PINP ELISA kitImmunodiagnostic SystemsAC-33F1
Commercial assay or kitNuclear/Cytosol Fractionation KitBiovisionK266
OtherFetal bovine serumSigma-AldrichF2442
OtherPenicillin Streptomycin 100x solutionHyCloneSV3001
OtherDMEM, low glucoseGibco11885084
OtherDMEM, high glucoseGibcoD5671
Author response table 1
RNA-seq analysis of LepR+ cells from non-irradiated bone marrow as well as hematopoietic cells and unfractionated cells from irradiated bone marrow (which are high in adipocytes).

Itga11 is poorly expressed in adipocyte-rich bone marrow cells after irradiation (all data represent average FPKM values from multiple replicates).

https://doi.org/10.7554/eLife.42274.024
Cell typeAdipoq Fabp4 Plin1 Itga11 Osteolectin
LepR+ cells from bone marrow835231.23437
Hematopoietic cells from irradiated bone marrow1.8130.30.10.2
Whole bone marrow cells after irradiation (high in adipocytes)921016682.22.8

Data availability

Source data files have been provided for all figures.

Additional files

Supplementary file 1

Markers used for the flow cytometric isolation of bone marrow cell populations.

Stem and progenitor cell populations were isolated from mouse bone marrow by flow cytometry using the listed markers. Lineage (Lin) markers used to isolate Lineage negative cell populations were CD2, CD3, CD5, CD8, Ter119, Gr-1, and B220.

https://doi.org/10.7554/eLife.42274.021
Transparent reporting form
https://doi.org/10.7554/eLife.42274.022

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