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LAP-tTA and TRE-MYC mice were previously described and MYC expression in the liver was activated by removing doxycycline treatment (100 μg ml−1) from the drinking water of 4-week-old double transgenic mice for both TRE-MYC and LAP-tTA as previously described9, 13. C57BL/6 mice were obtained from NCI Frederick. Chemically induced HCC was established by intraperitoneal injection of diethylnitrosoamine (DEN) (Sigma) into 2-week-old male pups at a dose of 20 μg g−1 body weight13. Twelve-week-old male B6.Cg-Lepob/J (ob/ob) mice or wild-type control mice were obtained from Charles River. Foxp3–GFP mice were previously described31. NAFLD was induced by feeding mice with a methionine–choline-deficient (MCD) diet (catalogue number 960439, MP biomedical), a choline-deficient and amino-acid-defined (CDAA) diet (catalogue number 518753, Dyets) or a high-fat diet (catalogue number F3282, Bio Serv) for the indicated time10, 11, 32. The MCD diet was supplied with corn oil (10%, w/w), and no fish oil was added. Control diet was purchased from MP Biomedical (catalogue number 960441). Custom-made high- or low-linoleic-acid mouse diets were purchased from Research Diets. The modified diets were based on AIN-76A standard mouse diet, and are isocaloric (4.45 kcal g−1) and contained the same high-fat content (23%, w/w). Linoleic-acid-rich safflower oil and saturated fatty-acid-containing coconut oil were supplied at different ratios to yield 2% (w/w) for the low-linoleic-acid diet or 12% (w/w) for the high-linoleic-acid diet. C57BL/6 mice were fed with the high- or low-linoleic-acid diet for 4 weeks. MYC mice were injected i.p. with 50 μg CD4 antibody (clone GK1.5, BioXcell) every week for the indicated time period to deplete CD4+ T cells33. N-acetylcysteine (NAC) was given in drinking water (10 mg ml−1)34 for the indicated time period to prevent excess ROS production. Mitochondrial-specific antioxidant mitoTEMPO was purchased from Sigma. Mice received mitoTEMPO at a dose of 0.7 mg kg−1 per day25 by osmotic minipumps (ALZET). At the experimental end points, mice were killed. For flow cytometry analysis, single-cell suspensions were prepared from spleen, liver and blood as described previously. Red blood cells were lysed by ACK Lysis Buffer (Quality Biologicals). Parts of live tissue were fixed by 10% formaldehyde and subjected to H&E staining. Free fatty acids were purchased from Sigma. Lipid accumulation was detected by Oil Red O staining in frozen liver sections using the custom service of Histo Serv. Cells were surface-labelled with the indicated antibodies for 15 min at 4 °C. Flow cytometry was performed on BD FACSCalibur or BD LSRII platforms and results were analysed using FlowJo software version 9.3.1.2 (TreeStar). The following antibodies were used for flow cytometry analysis: anti-CD3-FITC (clone 17A2, BD Pharmingen), anti-CD4-PE (clone RM4–4, Biolegend), anti-CD4-APC (clone RM4–5, eBioscience), anti-CD8-Alexa Fluor 700 (clone 53–6.7 Biolegend), anti-CD45, anti-CD44-PE (clone IM7, eBioscience), anti-CD62L-PerCP/Cy5.5 (MEL-14, Biolegend), anti-CD69-Pacific blue (clone H1.2F3, Biolegend), PBS57/CD1d-tetramer-APC (NIH core facility). To determine cytokine production, cells were stimulated with PMA and ionomycine for 30 min, and then were fixed and permeabilized using cytofix/cytoperm kit (BD Pharmingen) followed by anti-IFN-γ-PE (clone XMG1.2, BD Pharmingen), anti-IL-17-PerCP/Cy5.5 (clone TC11-18H10.1, Biolegend) staining. Cell death and apoptosis were detected with annexin V-PE (BD Pharmingen) and 7-AAD (BD Pharmingen) staining according to the manufacturer’s instructions. Intrahepatic CD4+ lymphocytes were gated on the CD3hiCD4+ population from total live hepatic infiltrating mononuclear cells. Absolute numbers were calculated by multiplying frequencies obtained from flow by total live mononuclear cell count, then divided by liver weight. The antibodies used for human peripheral blood mononuclear cell (PBMC) staining are the following: anti-CD3-PE (clone SK7, BD Pharmingen), anti-CD4-FITC (clone RPA-T4, BD Pharmingen), anti-CD8-APC (clone RPA-T8, BD Pharmingen). Murine T assays were performed as described31. Briefly, liver T cells were isolated as CD4+GFP+ by flow-cytometry-assisted cell sorting from Foxp3–GFP mice kept on an MCD or control diet for 4 weeks. CD4+GFP− T effector (T ) cells (5 × 104) were stimulated for 72 h in the presence of irradiated T-depleted splenocytes (5 × 104) plus CD3ε monoclonal antibody (1 μg ml−1), with or without T cells added at different ratios. 3H-Thymidine was added to the culture for the last 6 h and incorporated radioactivity was measured. Freshly isolated splenocytes from MYC-ON MCD mice were incubated with 5 μg ml−1 of mouse α-fetoprotein protein (MyBioSource) for 24 h. Golgiplug was added for the last 6 h. Then, cells were fixed and permeabilized using cytofix/cytoperm kit (BD Pharmingen) followed by anti-IFN-γ-PE (clone XMG1.2, BD Pharmingen) staining. Primary mouse hepatocytes were isolated from MYC mice and cultured according to a previous report35. Briefly, mice were anaesthetized and the portal vein was cannulated under aseptic conditions. The livers were perfused with EGTA solution (5.4 mM KCl, 0.44 mM KH PO , 140 mM NaCl, 0.34 mM Na HPO , 0.5 mM EGTA, 25 mM Tricine, pH 7.2) and Gey’s balanced salt solution (Sigma), and digested with 0.075% collagenase solution. The isolated mouse hepatocytes were then cultured with complete RPMI media in collagen-I-coated plates. Hepatic fatty acid composition was measured at LIPID MAPS lipidomics core at the University of California (San Diego) using an esterified and non-esterified (total) fatty acid panel. Briefly, liver tissues were homogenized and lipid fraction was extracted using a modified Bligh Dyer liquid/liquid extraction method. The lipids were saponified and the hydrolysed fatty acids were extracted using a liquid/liquid method. The extracted fatty acids were derivatized using pentaflourylbenzylbromine (PFBB) and analysed by gas chromatography (GC) using an Agilent GC/mass spectrometry (MS) ChemStation. Individual analytes were monitored using selective ion monitoring (SIM). Analytes were monitored by peak area and quantified using the isotope dilution method using a deuterated internal standard and a standard curve. Isolated primary hepatocytes from MYC mice fed with MCD or control diet were cultured in complete RPMI for 24 h. Supernatant were harvested and FFAs were identified by GC/MS. Splenocytes from MYC mice were cultured with or without 50 μM C18:2 for 24 h. CD4+ and CD8+ T lymphocytes were sorted and total RNA was extracted using miRNeasy mini kit (Qiagen). Array analysis was performed in the Department of Transfusion Medicine, clinical centre at NIH. Mouse gene 2.0 ST array (Affymetrix) was used and performed according to the manufacturer’s instruction. Data were log-transformed (base 2) for subsequent statistical analysis. The Partek Genomic Suite 6.4 was used for the identification of differentially expressed transcripts. The Ingenuity Pathway Analysis tool (http://www.ingenuity.com) was used for analysis of functional pathways. RNA was extracted from frozen tissues with RNeasyMini Kit (Qiagen). Complementary DNA was synthesized by iScriptcDNA synthesis kit (BioRad). Sequence of primers used for quantitative RT–PCR can be obtained from the authors. The reactions were run in triplicates using iQSYBR green supermix kit (BioRad). The results were normalized to endogenous GAPDH expression levels. CD4+ T lymphocytes were isolated from the spleen of MYC mice by negative autoMACS selection using a CD4+ T lymphocytes isolation kit (Miltenyi Biotec) or flow cytometry cell sorting. Human CD4+ T lymphocytes were prepared from PBMCs by autoMACS using a CD4+ T lymphocytes isolation kit (Miltenyi Biotec). The purity of CD4+ T lymphocytes was above 90% after autoMACS separation and above 95% after flow cytometry cell sorting. C16:0, C18:0, C18:1,and C18:2 were purchased from Sigma. Fatty acids were dissolved in DMEM with 2% fatty-acid-free bovine serum albumin (BSA; Sigma, catalogue number A8806) after solvent was evaporated, then followed by two rounds of vortexing and 30 s of sonication. Isolated CD4+ T lymphocytes or splenocytes were incubated with different fatty acids or conditioned medium from hepatocyte culture for 3 days. Unless specifically described, fatty acids were used at 50 μM concentration. For fatty acid depletion, active charcoal (catalogue number C-170, Fisher) was used as described before36. Briefly, 0.5 g of active charcoal was added into every 10 ml of conditioned medium. Then pH was lowered to 3.0 by addition of 0.2 N HCl. The solution was rotated at 4 °C for 2 h. Charcoal was then removed by centrifugation, and the clarified solution was brought back to pH 7.0 by addition of 0.2 N NaOH. NAC (10 mM), catalase (1,000 U ml−1) or mitoTEMPO (10 μM) was used to inhibit ROS production, mitochondrial ROS levels were determined by mitoSOX staining 24 h after treatment, cell death and apoptosis were measured by annexin V and 7-AAD staining 3 days after treatment. Caspase activity assay was measured by caspase-Glo 3/7 assay kit (Promega) according to the manufacturer’s protocol. Fresh prepared liver-infiltrating mononuclear cells were washed and resuspended in 500 μl of BODIPY 493/503 at 0.5 μg ml−1 in PBS. Cells were stained for 15 min at room temperature. Then cells were subjected to flow cytometry analysis. Two pZIP lentiviral shRNA vectors targeting human CPT1a and a control vector (NT#4) were purchased from TransOMIC Technologies. Lentivirus was packed in 293T cells. Jurkat cells were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), and no authentication test was performed by us. Cells were cultured in complete RPMI medium and were tested to be mycoplasma free. Jurkat cells were infected with shRNA lentivirus. Puromycin was added to eliminate non-transduced cells. Doxycycline (100 ng ml−1) was added to induce shRNA and GFP expression for 3 days. Efficiency of shRNAs was confirmed by western blot. Jurkat cells with CPT1a knockdown were treated with 200 μM C18:2 for 24 h. Mitochondrial ROS production and cell survival were measured in GFP+-transduced cells. Fatty acid oxidation was measured according to a previous publication37. 1-14C-C18:2 and 1-14C-C16:0 were purchased from PerkinElmer. Briefly, isolated CD4+ or CD8+ T lymphocytes were pretreated with C18:2 or kept in regular media. After 24 h, cell media was changed to media containing 50 μM cold C18:2 plus 1 μCi 1-14C-C18:2 per ml or 50 μM cold C16:0 plus 1 μCi 1-14C-C16:0 per ml. After 2 h, medium was removed and mixed with concentrated perchloric acid (final concentration 0.3 M) plus BSA (final concentration 2%) to precipitate the radiolabelled fatty acids. Samples were vortexed and centrifuged (10,000g for 10 min). Radioactivity was determined in the supernatant to measure water-soluble β-oxidation products. Mitochondrial membrane potential was measured by TMRM (ImmunoChemistry Technologies) staining according to the manufacturer’s protocol. Briefly, cells were kept in culture medium with 100 nM of TMRM for 20 min in a CO incubator at 37 °C. After washing twice, cells were processed to flow cytometry analysis. Mitochondria-associated superoxide was detected by mitoSOX (Life Technologies) staining according to the manufacturer’s protocol. Briefly, cells were first subjected to surface marker staining. Then cells were stained with 2.5 μM mitoSOX for 30 min in a CO incubator at 37 °C. After washing twice, cells were processed for flow cytometry analysis. OCR was measured using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience) as previously described38. AutoMACS-sorted mouse CD4+ and CD8+ T lymphocytes were attached to XFe96 cell culture plates using Cell-Tak (BD Bioscience) in RPMI media with 11 mM glucose. Cells were activated with 1:1 CD3:CD28 beads (Miltenyi BioTech) and vehicle or 50 μM C18:2 was added. Twenty-four hours after activation, cells were incubated in serum-free XF Base Media (Seahorse Bioscience) supplemented with 10 mM glucose, 2 mM pyruvate and 2 μM glutamine, pH 7.4, along with 50 μM C18:2 if previously present, for 30 min at 37 °C in a CO -free cell culture incubator before beginning the assay. Five consecutive measurements, each representing the mean of 8 wells, were obtained at baseline and after sequential addition of 1.25 μM oligomycin, 0.25 μM trifluorocarbonylcyanide phenylhydrazone (FCCP), and 1 μM each of rotenone and antimycin A (all drugs from Seahorse Bioscience). OCR values were normalized to cell number as measured by the CyQUANT Cell Proliferation Assay Kit (Life Technologies). Human liver samples were stained as previously described8. For immunostaining, formalin-fixed, paraffin-embedded human liver tissue samples were retrieved from the archives of the Institute of Surgical Pathology, University Hospital Zurich. Fibrosis grade was analysed for NASH according to NAFLD activity score (NAS)39 and for others according to METAVIR score40. The study was approved by the local ethics committee (Kantonale Ethikkommission Zürich, application number KEK-ZH-Nr. 2013-0382). Human PBMCs from healthy donors were obtained on an NIH-approved protocol and prepared as described previously41. Informed consent was obtained from all subjects. The sample sizes for animal studies were guided by a previous study in our laboratory in which the same MYC transgenic mouse stain was used. No animals were excluded. Neither randomization nor blinding were done during the in vivo study. However, mice from the same littermates were evenly distributed into control or treatment groups whenever possible. The sample size for the patient studies was guided by a recent publication also studying NASH-induced HCC, but focused on different aspects8. Statistical analysis was performed with GraphPad Prism 6 (GraphPad Software). Significance of the difference between groups was calculated by Student’s unpaired t-test, one-way or two-way ANOVA (Tukey’s and Bonferroni’s multiple comparison test). Welch’s corrections were used when variances between groups were unequal. P < 0.05 was considered as statistically significant.


The Axl−/− 31, Mertk−/− 31, Axl−/−Mertk−/− 31, Gas6−/− 31Pros1fl/flNesCre10, 32, Pros1fl/-NesCre10, Cx3cr1GFP/+ 22, Cx3cr1CreER 24, S100bGFP/+ 33 and SNCAA53T (ref. 6) strains have been described previously. The Mertkfl/fl mouse line diagrammed in Extended Data Fig. 4 was generated by inGenious Targeting Laboratory (iTL, Ronkonkoma NY), using iTL C57Bl/6 embryonic stem (ES) cells. This line targets exon 18, a 137 nucleotide (nt) sequence that encodes residues W779–L824 within the Mer kinase domain. Cre-mediated deletion of this exon introduces a frame shift and a stop codon one amino acid downstream of exon 17. This truncated, kinase-dead protein and/or its mRNA are apparently unstable, as antibodies directed against the Mer extracellular domain do not detect a truncated protein upon Cre-mediated excision (see text). Deletion of exon 18 therefore effectively generates a protein null. The complete Mertk mouse knockout31 deletes exon 17, a 160 nt sequence that encodes M725–V778 within the Mer kinase domain. (Exon 17 was numbered as exon 18 in the original description of the Mertk knockout allele31.) This single exon deletion also introduces a frame shift (five amino acids downstream of exon 16), produces an unstable protein, and also results in a Mer protein null12. The Neo cassette was removed via Flp-mediated recombination by crossing high-percentage chimaeric mice to C57Bl/6 FLP mice. Neo deletion was confirmed by PCR. These Mertkfl/fl mice, together with PCR-based protocols for their genotyping, are available upon request from the Rothlin laboratory (contact C.V.R.). Recombination (inactivation) of the Mertkfl/fl allele in Cx3cr1CreER/+Mertkfl/fl mice was achieved using tamoxifen injection. Cx3cr1CreER/+Mertkfl/fl mice (16 weeks) received a dose (150 mg kg−1 body weight) of tamoxifen (Sigma) as a solution in corn oil (Sigma) by intraperitoneal (i.p.) injection. Control mice received an i.p. injection of vehicle (corn oil) alone. Mice were analysed for Mer expression and apoptotic-cell (cCasp3+ cell) accumulation 1 week, 3 weeks or 7 weeks after injection. Mice analysed at 1 week received a single dose of tamoxifen or oil; mice analysed at 3 and 7 weeks received two successive injections 48 h apart. All lines, with the exception of the Mertkfl/fl alleles, have been backcrossed for >9 generations to a C57BL/6 background. All animal procedures were conducted according to protocols approved by the Salk Institute Animal Care and Use Committee (Protocol No. 11-00051). Mice (both males and females) were randomly allocated to experimental groups (three to six mice per group) and investigators were blinded to group allocation during the experiment. Investigators were not blinded to sample identity. Group size was based on previous literature. No statistical methods were used to predetermine sample size. Dexamethasone, 5-Bromo-2-deoxyuridine and DMSO were from Sigma-Aldrich. Poly(I:C) was from Invivogen. Lipopolysaccharide (LPS) (Escherichia coli serotype O55:B5) was from Enzo. IFN-γ was from BioVision. Purified human protein S was from Haematologic Technologies. Recombinant mouse Gas6 was produced as described previously5. Antibodies used were as follows: anti-Mer (AF591), anti-Axl (AF854), and anti-Gas6 (AF986) were from R&D Systems, anti-Mer (DS5MMER) from eBioscience, anti-Iba1 (019-19741) was from Wako, anti-GFAP (z0334) was from Dako, anti-Neurofilament H (SMI-31 NE1022), anti-NeuN (MAB377 A60), anti-Calretinin (AB1550), anti-Tyrosine Hydroxylase (MAB318; LNC1) and anti-GAPDH (MAB374; 6C5) were from Millipore, anti-α-synuclein (C-20-R sc-7011-R) and anti-Axl (M-20 sc-1097) were from Santa Cruz, anti-cCaspase 3 (Asp175) was from Cell Signaling, anti-ACSA-2 (clone IH3-18A3) was from Miltenyi Biotec, anti-CD169 (Siglec1; 3D6) and anti-BrdU (BU1/75 (ICR1) were from AbD serotec, and anti CD31 (ab28364) and anti-S100b (EP1576Y) were from Abcam. Secondary antibodies used for immunoblot analysis were horseradish-peroxidase-conjugated anti-goat (705-035-003) from Jackson ImmunoResearch, and anti-mouse (NA931V) and anti-rabbit (NA934V) from GE Healthcare. Secondary antibodies for immunocyto- and immunohistochemistry were fluorophore-conjugated anti-goat (A-11055 from Life Technologies, or 705-166-147 from Jackson ImmunoResearch), anti-rabbit (A-10040 or A-21206 from Life Technologies), and anti-mouse (A-11029 from Life Technologies, or 715-166-150 from Jackson ImmunoResearch). Adult mice (3–6 month) were anaesthetized with 2.5% avertin in saline, perfused with 20 U ml−1 heparin in PBS, and subsequently with 4% PFA in PBS. Brain and spinal cords were collected, immersion-fixed overnight at 4 °C, infiltrated with 30% sucrose in PBS overnight at 4 °C, and flash-frozen in tissue freezing medium. Sections of 17 μm were cut, air-dried overnight at room temperature and subsequently processed for staining. Non-specific binding was blocked by 1 h incubation in blocking buffer (PBS containing 0.1% Tween-20, 5% donkey serum and 2% IgG-free BSA). Sections were incubated overnight at 4 °C with primary antibody (identified above) diluted in blocking buffer, then washed in PBS 0.1% Tween-20, and incubated for 2 h at 22–24 °C in the dark with Hoechst and fluorophore-coupled secondary antibodies diluted in blocking buffer. Sections were washed, sealed with Fluoromount-G (SouthernBiotech) and stored at 4 °C. Images were acquired with a Zeiss LSM 710 confocal microscope using Plan-Apochromat 40× and 63× objectives. Cleaved Casp3+ apoptotic cells were counted in four successive 17 μm sections that spanned the SVZ in three different mice for both the Mertk−/− and Axl−/−Mertk−/− genotypes, and in two different mice for the Mertk−/−Gas6−/− genotype. No cCasp3+ cells in excess of wild-type were observed in SVZ sections of any of the other genotypes analysed. The cross-sectional area of the SVZ was defined as the region of intense Hoechst 33258 staining, as illustrated in Figs 1a, c, d, and measured using ImageJ. Accumulation of apoptotic cells between the Axl−/−Mertk−/− and Mertk−/−Gas6−/− genotypes is not statistically different. Note that cCasp3 marks a subset of apoptotic cells. Three successive injections (50 mg kg−1 body weight) of 5-bromo-2-deoxyuridine (BrdU) were performed in 8-week-old mice at 24 h intervals and BrdU staining was assessed 35 days later. Briefly, mice were anaesthetized with 2.5% avertin in saline, perfused with 20 U ml−1 heparin in PBS, and subsequently with 4% PFA in PBS. Brain were collected, immersion fixed overnight at 4 °C, infiltrated with 30% sucrose in PBS overnight at 4 °C and flash-frozen in tissue freezing medium. Sections of 17 μm were cut and air-dried overnight at room temperature. Subsequently, the sections were incubated in 2 N HCl at 37 °C for 30 min, rinsed for 10 min in 0.1 M borate buffer (pH 8.4) at room temperature and washed six times in PBS. To block endogenous peroxidase activity, sections were incubated for 10 min in 0.3% H O in 10% methanol. Non-specific binding was blocked by 1 h incubation in blocking buffer (PBS containing 0.25% Triton-X and 5% donkey serum). Sections were incubated for 72 h at 4 °C with primary antibody (anti-BrdU) diluted in blocking buffer, then washed in PBS 0.1% Tween-20, and incubated for 2 h at room temperature in the dark with a biotin-conjugated secondary antibody diluted in blocking buffer. Sections were washed and 3,3′-diaminobenzidine (DAB) staining was performed using Vectastain Elite ABC-kit (Vector Laboratories) and DAB peroxidase (HRP) substrate kit (Vector Laboratories) following manufacturer’s instructions. Afterwards, sections were counterstained using haematoxylin for 15 s, sealed with Vectamount (Vector Laboratories) and stored at room temperature. Images were acquired with a Zeiss slide scanner Axio Scan.Z1 using 20× objective and analysed with ImageJ. For quantitation, BrdU+ cells in granule cell layer and glomerular layer of the olfactory bulb were counted in two consecutive sections per animal and averaged per animal. Cells were fixed for 10 min in 4% PFA/4% sucrose in PBS, washed with PBS, incubated for 10 min in 100 mM glycine, permeabilized for 5 min in 0.2% Triton-X100 in PBS, washed with PBS, and nonspecific binding was then blocked by 40 min incubation in blocking buffer (2% IgG-free BSA in PBS). Coverslips were incubated for 1 h at 22–24 °C with primary antibody diluted in blocking buffer, washed five times in PBS, and then incubated for 1 h at 22–24 °C in the dark with Hoechst stain and fluorophore-coupled donkey secondary antibody (identified above) diluted in blocking buffer. Coverslips were washed and mounted on slides with Fluoromount-G (SouthernBiotech) and stored at 4 °C. Images were acquired with a Zeiss LSM 710 confocal microscope using Plan-Apochromat 40× and 63× objectives. One cerebral hemisphere from a Cx3cr1GFP/+ mouse was cleared using CLARITY protocols, essentially as described34. Rather than electrophoretic clearing, samples were incubated at 37 °C and passively cleared over 3 weeks by daily replacement of the clearing solution. A 1 mm3 block of tissue adjacent to the lateral ventricle of Cx3cr1GFP/+ mice, in the region containing the SVZ, was imaged using a Zeiss LSM 710 confocal microscope. Fiji software was used to assemble images. Cultured cells were washed with ice-cold DPBS and lysed on ice in 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1% Triton-X100, 0.27 M sucrose, and protease and phosphatase inhibitors (Roche). Tissues were snap-frozen in liquid nitrogen before lysis. For immunoblot analysis, equal amounts of protein in LDS sample buffer (Invitrogen) were separated by electrophoresis through 4–12% Bis-Tris polyacrylamide gels (Novex, Life Technologies) and transferred to PVDF membranes (Millipore). For Axl immunoprecipitation, tissue lysates were precleared overnight at 4 °C with Protein G-Sepharose (Invitrogen). This was then removed and lysates were incubated for 2 h with 0.2 μg anti-Axl (M20) for 0.5 mg protein in cell lysate. Fresh Protein G–Sepharose was added for 2 h and immunoprecipitates were washed twice with 1 ml of lysis buffer containing 0.5 M NaCl and once with 1 ml of 50 mM Tris-HCl (pH 7.5). Immunoprecipitates were eluted in LDS buffer, separated by electrophoresis through polyacrylamide gels and transferred to PVDF membranes. Nonspecific binding was blocked with TBST (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl and 0.1% Tween-20) containing 5% BSA, and membranes were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. Blots were then washed in TBST and incubated for 1 h at 22–24 °C with secondary horseradish peroxidase–conjugated antibodies in 5% skim milk in TBST. After repeating the washes, signal was detected with enhanced chemiluminescence reagent. Total cellular RNA was isolated with an RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen). DNA was removed by on-column digestion with DNase (Qiagen). An RT Transcriptor First Strand cDNA Synthesis Kit (Roche) with anchored oligonucleotide (dT) primers (Roche) was used for reverse transcription. Quantitative PCR was run in a 384-well plate format on a ViiA 7 Real-Time PCR System (Applied Biosystems) with 2 × SYBR Green PCR Master Mix (Applied Biosystems). Primers are listed in Supplementary Table 1. Expression was analysed by the threshold cycle (∆ΔC ). Postnatal day 30 (P30) to P50 mice (Cx3cr1GFP/+, Axl−/−Mertk−/−Cx3cr1GFP/+) brains were dissociated using Neural Dissociation kit, Postnatal Neurons and the gentleMACS dissociator according to the manufacturer’s instructions (Miltenyi). Single cell suspensions were resuspended in 30% Percoll in HBSS solution and centrifuged 15 min at 700 g to remove myelin. Cells were grown for 7 days in DMEM-F12 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin before being processed for immunostaining or phagocytosis assay. Cytosine β-D-arabinofuranoside (Ara-C, 5 μM) was added after 5 days in vitro to limit fibroblast proliferation. When astrocytes were also isolated, microglia were first purified using C11b MicroBeads (Miltenyi) and grown for 7 days in DMEM-F12 with 10% FBS and 1% penicillin/streptomycin while astrocytes were grown for 10 days in MACS Neuro Medium with 2% MACS NeuroBrew-21 and 1% penicillin/streptomycin. For the generation of apoptotic cells, thymocytes were isolated from 3- to 6-week-old mice, red blood cells were lysed with ACK buffer and remaining cells were incubated for 6 h in RPMI medium containing 5% FBS and 2 μM dexamethasone to induce apoptosis. This routinely resulted in 70% apoptotic and ≤5% necrotic cells. Apoptotic cells were then stained for 30 min with100 ng ml−1 pHrodo-s.e. (Invitrogen) as described previously12, 35, 36. Labelled cells were washed twice in PBS containing 1% BSA (to block remaining pHrodo-SE) and 1 mM EDTA (to remove any bound Gas6 and protein S) and once with DMEM. Apoptotic cells were then incubated for 10 min with recombinant mouse Gas6 or purified human protein S, added to microglia or astrocyte cultures at a ratio of 10:1 (apoptotic cells:phagocytes), and incubated for 1 h at 37 °C. Microglia or astrocytes were then briefly washed in DPBS, incubated for 10 min at 37 °C in trypsin (0.25%), and then placed on ice and detached by vigorous pipetting. Astrocytes were labelled using anti-ACSA2-APC antibody37. Phagocytosis was assessed by flow cytometry with post-acquisition data analysis with FlowJo software (TreeStar). pHrodo fluorescence was measured with excitation at 561 nm and emission filters for phycoerythrin (574–590 nm) on a LSR II (BD Biosciences) at the Flow Cytometry Core of the Salk Institute, as described previously12. Microglia were gated as GFP+ cells and astrocytes were gated as APC+ cells. Adult male mice (3–6 months old) were anaesthetized with isoflurane (1.5–2.5% in 100% oxygen at 0.8–1.0 l min−1). Body temperature was kept at 36–37 °C, and hydration status was maintained using subcutaneous physiological saline injections (0.1 ml per 25 g body weight every 1–2 h). For head plate implantation, hair, skin and periosteum overlying the neocortex were removed. After cleaning exposed skull areas, a custom metal head plate was affixed to the skull using OptiBond (31514; Kerr) and dental acrylic (H00335; Coltene Whaledent), keeping the intended imaging area over somatosensory or visual cortex uncovered. A polished and reinforced thinned skull window (~2–3-mm diameter; ~20–50 μm remaining bone thickness) was then prepared, as described previously38, 39. A movable objective microscope (Sutter Instrument) equipped with a pulsed femtosecond Ti:Sapphire laser (Chameleon Vision II or Ultra II, Coherent), two fluorescence detection channels (565DXCR dichroic, ET525/70M-2P and ET605/70M-2P emission filters, Chroma; H7422-40 GaAsP photomultiplier tubes, Hamamatsu), and a water immersion objective (LUMPlanFL N 40XW 0.8NA; Olympus) was used for two-photon imaging. Imaging was performed as described previously28, 39 using 920–940 nm centre excitation wavelengths. Average laser powers used for transcranial optical recordings depended on imaging depth (typically ~10–30 mW at ~150–200 μm depth from the pia). Images were typically acquired using a 6 Hz frame rate, 256 × 256 pixel resolution and a 5-frame average. Image stacks were acquired every 1.5–2 min for up to 5 h and typically contained 20–30 images per stack with 1 μm axial image spacing. Fields-of-view had a typical side length of 65–100 μm. Imaging settings were kept constant during time-lapse recordings. For quantitative image data analysis, ImageJ or Fiji software was used. First, maximum intensity images were produced from individual image stacks. Then, lateral image shifts in time-lapse recordings were corrected using a custom-written ImageJ alignment plugin based on the position shift of the peak in cross-correlation images, typically using the first projection image as the reference image. Structural dynamics of individual microglial cell processes was quantified manually using the MTtrackJ plugin in Fiji. Image analysis was done blind with respect to experimental condition. Videos were also created with Fiji. To target blood vessels for focal laser lesion, blood plasma was stained by tail vein injection of biocytin-TMR (2–5% in saline, T-12921, Life Technologies). Lesions were performed following a baseline recording period of 30–45 min, during which z-stacks were acquired as described above. To induce lesions, the Ti:Sapphire laser was transiently tuned to 800 nm and a confined area (8–15 μm diameter, ~1 μm axial extent) of a horizontally oriented cortical capillary at 150–220 μm depth was exposed to 70–130 mW for 10–30 s. Laser lesions caused extravasation of dye, indicating disruption of the blood-brain barrier. Following focal lesion, image stack acquisition was resumed using the same laser and recording parameters as during the baseline recording period. Although Supplementary Videos 4 and 5 run for only ~12 min (the time required for microglial processes to reach the lesion site), time-lapse recording of the same cortical volume continued for 2–4 h after the lesion.


C57BL/6J males were used for analysis of wild-type bone unless stated otherwise. Mice at the age of 2–5 weeks and 55–70 weeks were chosen for young and aged group sets, respectively. All EC-specific mutants were generated using Cdh5(PAC)-CreERT2 transgenic mice unless indicated otherwise. For gene inactivation in the postnatal endothelium, mice carrying loxP-flanked Rbpj (Rbpjlox/lox) alleles31 and Cdh5(PAC)-CreERT2 transgenics32 were interbred. To induce Cre activity and gene inactivation, offspring were injected with 500 µg tamoxifen (Sigma, T5648) intraperitoneally every day from postnatal day (P)10 to P14. The resulting RbpjiΔEC (CreERT2T/+ Rbpjlox/lox) mutants and Cre-negative littermate controls were killed at P28, and femurs and tibiae were collected for analysis. Identical breeding and tamoxifen administration strategies were used to generate EC-specific mutants with Fbxw7lox/lox (ref. 33) or Dll4lox/lox mice34. For EC-specific Hif1a deletions, Cdh5(PAC)-CreERT2 transgenic mice were interbred with conditional Hif1a (Hif1alox/lox) mutants35. To induce Cre activity and gene inactivation, pups were injected with 500 µg tamoxifen (Sigma, T5648) intraperitoneally every day from P10 to P14. Femurs and tibiae from Cdh5(PAC)-CreERT2T/+ Hif1alox/lox (Hif1aiΔEC) mutants and Cre-negative Hif1alox/lox (Controls) were collected on P20 after euthanasia. The same approach was used for experiments involving conditional Vhl mice36. For Fbxw7 deletion in the vasculature of aged mice, we generated litters with Fbxw7lox/lox Cdh5(PAC)-CreERT2T/+ (Fbxw7iΔEC) and Fbxw7lox/lox (control) genotypes. To induce Cre activity and gene inactivation, 55- to 65-week-old mice were injected with 1,000 µg tamoxifen (Sigma, T5648) intraperitoneally every day for 5 days. After a rest period of 16 days, mice were subjected to a second round of tamoxifen injections with the same dosage and frequency as described above. After a further 16 days, mice were analysed after euthanasia. For overexpression of the Notch1 intracellular domain (NICD), Gt(ROSA)26Sortm1(Notch1)Dam/J mice37 and Cdh5(PAC)-CreERT2 transgenics were interbred. Tamoxifen administration (see above for injection schedule) was used to generate CreERT2-positive (NICDiOEC) mutants overexpressing NICD in ECs and corresponding controls. For EC-specific PDGFB overexpression, Rosa26-hPDGF-B mice38 were interbred with Cdh5(PAC)-CreERT2 or with Tie2 Cre39 transgenics. To study the interplay between Notch and HIF signalling in ECs, endothelial specific double-mutant mice were generated using Cdh5(PAC)-CreERT2 transgenics. Cdh5(PAC)-CreERT2 mice were interbred with mice carrying the indicated combinations of Hif1alox/lox, Rbpjlox/lox, Vhllox/lox and Gt(ROSA)26Sortm1(Notch1)Dam/J alleles. For the detection of Notch cleavage and activity, Notch1tm3(cre)Rko/J (NICD-Cre) mice20, which carry a Cre recombinase fused to the carboxy (C) terminus of the intracellular domain of Notch1, were mated with Rosa26-mG/mT reporter animals21. The resulting Notch1tm3(cre)Rko/JT/+ R26-mG/mTT/+ double heterozygotes were killed and analysed at 3 weeks of age. Genetic labelling of cells expressing ephrin-B2 was performed using B6;129S4-Efnb2tm2Sor/J (Efnb2GFP/+) knock-in mice, which express H2B–GFP under control of the endogenous Efnb2 promoter8. For labelling of proliferating cells, mice were intraperitoneally injected with 300 µg of EdU (Invitrogen) 3 h before euthanasia. Tibiae were immediately collected and processed. BM cells and bone sections were stained for EdU using Click-iT chemistry following the manufacturer’s instructions (Invitrogen). For metabolic labelling with the hypoxia probe pimonidazole (Pimo, Hypoxyprobe), mutant and control mice were intraperitoneally injected with 60 mg/kg body weight Pimo at 2 h before euthanasia. Metabolized Pimo was detected by a rabbit antiserum against the non-oxidized, protein-conjugated form of pimonidazole (Hypoxyprobe). All animals were genotyped by PCR. Protocols and primer sequences can be provided upon request. Experiments involving animals were performed according to the institutional guidelines and laws, following protocols approved by local animal ethics committees. Freshly dissected bone tissues collected from wild-type mice or from mutants and their control littermates were immediately fixed in ice-cold 4% paraformaldehyde solution for 4 h. Decalcification was performed with 0.5 M EDTA at 4 °C with constant shaking and decalcified bones were immersed into 20% sucrose and 2% polyvinylpyrrolidone (PVP) solution for 24 h. Finally, the tissues were embedded and frozen in 8% gelatin (porcine) in presence of 20% sucrose and 2% PVP. For immunofluorescent stainings and morphological analyses, sections were generated using low-profile blades on a Leica CM3050 cryostat. For phenotypic analysis, mutant and littermate control samples were always processed, sectioned, stained, imaged and analysed together at the same conditions and settings. For immunostaining, bone sections were air-dried, permeabilized for 10 min in 0.3% Triton X-100, blocked in 5% donkey serum at room temperature for 30 min and probed with the primary antibodies diluted in 5% donkey serum in PBS for 2 h at room temperature or overnight at 4 °C. After primary antibody incubation (Supplementary Table 1), sections were washed with PBS three times and incubated with appropriate Alexa Fluor-coupled secondary antibodies (1:400, Molecular Probes) for 1 h at room temperature. Nuclei were counterstained with DAPI. Sections were thoroughly washed with PBS before mounting them using FluoroMount-G (Southern Biotech). Finally, cover slips were sealed with nail polish. Immunostaining of sorted HSCs and progenitor cells was performed as described previously40. Briefly, cells were pipetted onto poly-lysine coated slides, incubated for 10 min, fixed with 4% PFA for 10 min at room temperature, permeabilized in 0.15% Triton X-100 for 2 min at room temperature and blocked in 2% donkey serum overnight at 4 °C. Slides were then incubated for 2 h with the anti-phospho-H2AX, washed thrice and incubated with the appropriate secondary antibody. Immunofluorescent stainings were analysed at high resolution with a Zeiss laser scanning confocal microscope, LSM-780. Z-stacks of images were processed and reconstructed in three dimensions with Imaris software (version 7.00, Bitplane). Imaris, Photoshop and Illustrator (Adobe) software were used for image processing in compliance with Nature’s guidlines for digital images. All quantifications were done with ImageJ and Imaris software on high-resolution confocal images. For the analysis of messenger RNA (mRNA) expression levels in type H or type L endothelium, CD31hi Emcnhi and CD31lo Emcnlo cells were sorted by FACS directly into the lysis buffer of the RNeasy Mini Kit (QIAGEN). Total RNA was isolated according to the manufacturer’s protocol. A total of 100 ng RNA per reaction was used to generate complementary DNA (cDNA) with the iScript cDNA Synthesis System (Bio-Rad). qPCR was performed using TaqMan gene expression assays on an ABI PRISM 7900HT Sequence Detection System. The FAM-conjugated TaqMan probes Efnb2 and Sox17 were used along with TaqMan Gene Expression Master Mix (Applied Biosystems). Gene expression assays were normalized to endogenous VIC-conjugated Actb probes as standard. For analysis of mRNA expression levels from whole bones, dissected femurs or dissected metaphysis (as described in the figure legends) were immediately crushed finely, digested with collagenase and centrifuged to obtain a pellet, which was then lysed into lysis buffer of RNeasy Mini Kit (QIAGEN). For cells in culture, culture medium was completely removed and cells were immediately lysed with lysis buffer. A total of 500 ng RNA per reaction was used to generate cDNA with the iScript cDNA Synthesis System (Bio-Rad), which was further processed as described above. FAM-conjugated TaqMan probes Sp7, Cspg4, Pdgfrb, Sp7, Acan, Cfd, Hif1a, Epas1, Cxcl12, Fgf1, Kitl, Tgfb3, Tgfb1 and Vegfa were used along with TaqMan Gene Expression Master Mix (Applied Biosystems) to perform qPCR. For flow cytometric analysis and sorting of type H and type L ECs, tibiae and femurs were collected, cleaned thoroughly to remove the adherent muscles. The epiphysis was removed and only the metaphysis and diaphysis regions were processed. Tibiae were then crushed in ice cold PBS with mortar and pestle. Whole BM was digested with collagenase incubation at 37 °C for 20 min. Equal number of cells were then subjected to immunostaining with Emcn antibody (Santa Cruz, sc-65495) for 45 min. After washing, cells were stained with APC-conjugated CD31 antibody (R&D Systems, FAB3628A) for 45 min and phycoerythrin-conjugated secondary anti-rat antibody. After washing, cells were acquired on a BD FACS Canto flow cytometer or BD FACSVerse and analysed using BD FACSDiva (version 6.0, BD Bioscience) or BD FACSuite software. Cell sorting was performed with a BD FACS Aria II. For demarcating and sorting CD31hi Emcnhi ECs, first standard quadrant gates were set. Subsequently, to differentiate CD31hi Emcnhi cells from the total double positive cells in quadrant 2, gates were arbitrarily set at >104 log(Fl-4 (CD31-APC) fluorescence) and >104 log(Fl-2 (endomucin-PE) fluorescence). For the analysis of total ECs in bone, tibiae were processed as described above to obtain single-cell suspensions, which were stained with biotin-coupled CD45 (BD, 553077) or Ter119 (BD, 559971) antibodies for 45 min. After washing in PBS, cells were stained with Streptavidin PE-Cy5 (BD, 554062) and Alexa Fluor488-conjugated CD31 (R&D Systems, FAB3628G) antibodies for 45 min. After washing, cells were acquired on FACS Canto and FACS Verse flow cytometers and analysed using FACSDiva (version 6.0, BD Bioscience) and FACS Suite software respectively. Total bone ECs were quantified as CD31+/CD45−/Ter119−. Endomucin was used to distinguish Emcn− arterial ECs from Emcn+ sinusoidal and venous cells. For the analysis of HSC frequency in the BM, BM cells were isolated by crushing the long bones with mortar and pestle in Ca2+- and Mg2+-free PBS 2% heat-inactivated bovine serum. The cells were drawn by passing through a 25-gauge needle several times and filtered with a 70-μm filter. The following antibodies were used to stain HSCs: biotin-labelled lineage markers (CD5, CD11b, CD45R, Gr-1 and Ter119), cKit, Sca-1, CD48 and CD150 antibodies (Supplementary Table 1). For the enrichment and sorting of HSCs and progenitor cells, BM cells were isolated by crushing the long bones with mortar and pestle in Ca2+- and Mg2+-free PBS 2% heat-inactivated bovine serum. The cells were drawn by passing through a 25-gauge needle several times and filtered with a 70-μm filter. The obtained single-cell suspension obtained was subjected to lineage depletion (MACS, Miltenyi Biotech). Lineage-depleted BM cells were then stained with cKit and Sca1 antibodies (Supplementary Table 1). After washing, cell sorting was performed with a BD FACS Aria II. For the analysis of perivascular cells, Sca1+ ECs, ephrin-B2+ ECs and HSC frequency, the above-described protocol was used to obtain a single-cell suspension followed by immunostaining with the appropriate antibodies (Supplementary Table 1). Competitive repopulation assays were performed using the CD45.1/CD45.2 congenic system. Equivalent volumes of BM cells collected from EC-specific mutant mice or littermate control mice (CD45.2) were transplanted into lethally irradiated (12 Gy) CD45.1 recipients with 0.3 × 106 competitor CD45.1 cells. CD45.1/CD45.2 chimaerism of recipient blood was analysed up to 7 months after transplantation using flow cytometry analysis. For the secondary transplantation (performed for Fbxw7iΔEC mutant mice and littermate controls), 1 × 106 BM cells from CD45.1 mice that had previously undergone transplantation at 1:1 ratio were isolated at 7 months after transplantation and injected into lethally irradiated recipients. For calculation of competitive repopulating units (CRU), recipient mice were transplanted with limiting dilutions of donor-derived BM cells (2.5 × 104 to 2 × 105) together with 2 × 105 recipient-derived BM cells. Mice were killed after 18 weeks and the multi-lineage myelo-lymphoid donor-derived contribution in the peripheral blood was assessed using flow cytometry analysis. HSC-CRU frequency and statistical significance was determined using ELDA software (http://bioinf.wehi.edu.au/software/elda/)41, 42. Tibiae and femurs from wild-type mice were collected in sterile Ca2+- and Mg2+-free PBS, crushed with mortar and pestle, subjected to collagenase digestion, and filtered and washed thrice to obtain a single-cell suspension. Endothelial cells were then sorted using endomucin antibody (catalogue number SC-65495) and Dynabeads sheep anti-Rat IgG (Invitrogen). Sorted ECs were then plated on dishes coated with fibronectin and cultured in endothelial cell growth medium (EBM-2, Clonetics; Lonza) supplemented with EGM-2 SingleQuots (CC-4176, Clonetic; Lonza). PDGFRβ+ cells were sorted from single-cell suspensions using CD140b/PDGFB Receptor β antibody (eBioscience, catalogue number 14-1402-82) and Dynabeads sheep anti-Rat IgG (Invitrogen). Sorted PDGFRβ+ cells were cultured on tissue culture plates containing in alpha MEM (Gibco) and 10% fetal bovine serum (Gibco). Cultures of ECs or PDGFRβ+ cells were maintained at 37 °C with 5% CO in a humidified atmosphere. For DFM treatment and subsequent analysis, cultures between passage 1 and 2 were used. Cells were treated with DFM (6.25 mg ml−1 of culture medium) for the duration of 36 h and subsequently cell culture medium (directly) or cells after trypsinization, three washings and lysate preparation were used for ELISA. SCF levels in the mice bone supernatant/extracellular fluid (secreted SCF) or cell culture medium (secreted/extracellular SCF) or cell lysate from cultured cells (cellular/membrane-bound SCF) or cell lysate (cellular/membrane-bound SCF) prepared from the single-cell suspension of femurs (see primary culture section) were determined by ELISA kits (Sigma-Aldrich and USCN Business). CFU-F assay was performed as described previously16. MSC differentiation into osteogenic, chondrogenic and adipogenic lineages was performed using a MSC functional identification kit (R&D Systems) according to the manufacturer’s instructions. Fourteen days after differentiation, we performed qPCR analysis. All data are presented as either mean ± s.e.m. or mean ± s.d. (as indicated in figure legends). The data presented in the figures reflect multiple independent experiments performed on different days using different mice. Unless otherwise mentioned, most of the data presented in figure panels are based on three independent experiments. The significance of difference was determined using a two-tailed Student’s t-test unless otherwise mentioned. P > 0.05 was considered not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t-test with Welch’s correction was performed when group sizes were not equal. For analysis of the statistical significance of differences between more than two groups, we performed repeated measures one-way analysis of variance (ANOVA) tests with Greenhouse–Geisser correction (variances between groups were not equal) and Tukey’s multiple comparison tests to assess statistical significance with a 95% confidence interval. In all the figures, n refers to the number of mice. For data with unequal group sizes, the first numerical value for n refers to mutants and the second refers to control mice. All statistical analyses were performed using Graphpad Prism software. No randomization or blinding was used and no animals were excluded from analysis. Sample sizes were selected on the basis of previous experiments. Unless otherwise indicated, results are based on three independent experiments to guarantee reproducibility of findings.


No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were blinded to allocation of mice for assessment of histopathology and readouts of inflammation. E. coli strains were routinely cultured aerobically at 37 °C in lysogeny broth (LB) and on LB agar plates. B. abortus was cultured in tryptic soy broth or on tryptic soy agar (TSA) plates,. Chlamydia muridarum strain Nigg II was purchased from ATCC (Manassas, VA). Bacteria were cultured in HeLa 229 cells in DMEM supplemented with 10% FBS. Elementary bodies (EBs) were purified by discontinuous density gradient centrifugations as described previously23 and stored at −80 °C. The HEK293 cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS at 37 °C in a 5% CO atmosphere. HEK293 cells (ATCC CRL-1573) were obtained from ATCC and were grown in a 48-well tissue culture plates in DMEM containing 10% FBS until ~40% of confluency was reached. HEK293 cells were transfected with a total of 250 ng of plasmid DNA per well, consisting of 25 ng of the reporter construct pNF-κB-luc, 25 ng of the normalization vector pTK-LacZ, and 200 ng of the different combinations of mammalian expression vectors carrying the indicated gene (empty control vector, pCMV-HA-VceC5, pCMV-HA-TRAF2DN (this study), hNOD1-3×Flag, hNOD2-3×Flag, pCMV-HA-hRip2, hNOD1DN-3×Flag, hNOD2DN-3×Flag or pCMV-HA-Rip2DN24 and pCMV-myc-CDC42DN25. The dominant-negative form of TRAF2, lacking an amino-terminal RING finger domain26, was PCR amplified from cDNA prepared from HEK293 cells and cloned into the mammalian expression vector pCMV-HA (BD Biosciences Clontech). Forty-eight hours after transfection, cells were lysed either without any treatment, or stimulated with C12-iE-DAP (1,000 ng ml−1, InvivoGen) and MDP (10 μg ml−1, InvivoGen). After five hours of treatment the cells were lysed and analysed for β-galactosidase and luciferase activity (Promega). FuGene HD (Roche) was used as a transfection reagent according to the manufacturer’s instructions. Cell lines were monitored for mycoplasma contamination. Bone-marrow-derived macrophages (BMDMs) were differentiated from bone marrow precursors from femur and tibiae of C57BL/6 mice obtained from The Jackson Laboratory (Bar Harbor, ME), Nod1+/−Nod2+/− (wild-type littermates) and Nod1−/−Nod2−/− (NOD1/NOD2-deficient) mice (generated at UC Davis) as described previously27. For BMDM experiments, 24-well microtitre plates were seeded with macrophages at a concentration of 5 × 105 cells per well in 0.5 ml of RPMI media (Invitrogen, Grand Island, NY) supplemented with 10% FBS and 10 mM l-glutamine (complete RPMI) and incubated for 48 h at 37 °C in 5% CO . BMDMs were stimulated with C12-iE-DAP (1,000 ng ml−1, InvivoGen), MDP (10 μg ml−1, InvivoGen), thapsigargin (1 μM and 10 μM, Sigma-Aldrich), dithiothreitol (DTT) (1 mM, Sigma-Aldrich), and LPS (10 ng ml−1, InvivoGen) with or without pre-treatment (30 min) of the cells with IRE1α kinase inhibitor KIRA6 (1 μM, Calbiochem), IRE1α endonuclease inhibitor STF-083010 (50 μM, Sigma-Aldrich), PERK inhibitor GSK2656157 (500 nM, Calbiochem) and tauroursodeoxycholate TUDCA (200 μM, Sigma-Aldrich) in the presence of 1 ng ml−1 of recombinant mouse IFNγ (BD Bioscience, San Jose, CA). After 24 h of stimulation, samples for ELISA and gene expression analysis were collected as described below. Preparation of the B. abortus wild-type strain 2308 and the ∆vceC mutant inoculum and BMDM infection was performed as previously described27. Approximately 5 × 107 bacteria in 0.5 ml of complete RPMI were added to each well containing 5 × 105 BMDMs. Microtitre plates were centrifuged at 210g for 5 min at room temperature in order to synchronize infection. Cells were incubated for 20 min at 37 °C in 5% CO , and free bacteria were removed by three washes with PBS, and the zero-time-point sample was taken as described below. After the PBS wash, complete RPMI plus 50 mg ml−1 gentamicin and 1 ng ml−1 of recombinant mouse IFNγ (BD Bioscience, San Jose, CA) was added to the cells, and incubated at 37 °C in 5% CO . For cytokine production assays, supernatant for each well was sampled at 24 h after infection. In order to determine bacterial survival, the medium was aspirated at the time point described above, and the BMDMs were lysed with 0.5 ml of 0.5% Tween 20, followed by rinsing each well with 0.5 ml of PBS. Viable bacteria were quantified by serial dilution in sterile PBS and plating on TSA. For gene expression assays, BMDMs were suspended in 0.5 ml of TRI-reagent (Molecular Research Center, Cincinnati) at the time points described above and kept at −80 °C until further use. At least three independent assays were performed with triplicate samples, and the standard error of the mean for each time point was calculated. All mouse experiments were approved by the Institutional Animal Care and Use Committees at the University of California, Davis, and were conducted in accordance with institutional guidelines. Sample sizes were determined based on experience with infection models and were calculated to use the minimum number of animals possible to generate reproducible results. C57BL/6 wild-type mice and Rip2−/− mice (The Jackson Laboratory), Nod1+/−Nod2+/− (wild-type littermates) and Nod1−/−Nod2−/− (NOD1/NOD2-deficient) mice (generated at UC Davis) were injected intraperitoneally (i.p.) with 100 μl of 2.5 mg per kg body weight of thapsigargin (Sigma-Aldrich) at 0 and 24 h, and 4 h after the second injection the mice were euthanized and serum and tissues collected for gene expression analysis and detection of cytokines. Where indicated, mice were treated i.p. at 12 h before the first thapsigargin dose and 12 h before the second thapsigargin dose with the ER stress inhibitor TUDCA (250 mg per kg body weight). Female and male C57BL/6, Nod1+/−Nod2+/−, Nod1−/−Nod2−/− mice, and Rip2−/− mice aged 6–8 weeks, were held in micro-isolator cages with sterile bedding and irradiated feed in a biosafety level 3 laboratory. Groups of five mice were inoculated i.p. with 0.2 ml of PBS containing 5 × 105 CFU of B. abortus 2308 or its isogenic mutant ∆vceC, as previously described28. At 3 days post-infection, mice were euthanized by CO asphyxiation and their serum and spleens were collected aseptically at necropsy. The spleens were homogenized in 2 ml of PBS, and serial dilutions of the homogenate were plated on TSA for enumeration of CFU. Spleen samples were also collected for gene expression analysis as described below. When necessary, mice were treated i.p. at day one and two post-infection with a daily dose of 250 mg per kg body weight of the ER stress inhibitor TUDCA (Sigma-Aldrich), or 10 mg per kg body weight of the IRE1α kinase inhibitor KIRA6 (Calbiochem) or vehicle control. For the placentitis mouse model, C57BL/6, Nod1+/−Nod2+/− and Nod1−/−Nod2−/− mice, aged 8–10 weeks, were held in micro-isolator cages with sterile bedding and irradiated feed in a biosafety level 3 laboratory. Female Nod1+/−Nod2+/− mice were mated with male C57BL/6 mice (control mice) and female Nod1−/−Nod2−/− mice were mated with male Nod1−/−Nod2−/− mice (NOD1/NOD2-deficient), and pregnancy was confirmed by presence of a vaginal plug. At 5 days of gestation, groups of pregnant mice were mock infected or infected i.p. with 1 × 105 CFU of Brucella abortus 2308 or its isogenic mutant ∆vceC (day 0). At 3, 7 and 13 days after infection mice were euthanized by CO asphyxiation and the spleen and placenta of dams were collected aseptically at necropsy. At day 13 after infection (corresponding to day 18 of gestation), viability of pups was evaluated based on the presence of fetal movement and heartbeat, and fetal size and skin colour. Fetuses were scored as viable if they exhibited movement and a heartbeat, visible blood vessels, bright pink skin, and were of normal size for their gestational period. Fetuses were scored as non-viable if fetal movement, heartbeat, and visible blood vessels were absent, skin was pale or opaque, and their size for gestational period or compared to littermates was small, or they showed evidence of fetal reabsorption. Percentage of viability was calculated as [(number viable pups per litter/total number pups per litter) × 100]. At each time point, the placenta samples were collected for bacteriology, gene expression analysis and blinded histopathological analysis (Extended Data Fig. 6d). When indicated, mice were treated i.p. at days 5, 7 and 9 post-infection with a daily dose of 250 mg per kg body weight of the ER stress inhibitor tauroursodeoxycholate TUDCA (Sigma-Aldrich) or vehicle control. RNA was isolated from BMDMs and mouse tissues using Tri-reagent (Molecular Research Center) according to the instructions of the manufacturer. Reverse transcription was performed on 1 μg of DNase-treated RNA with Taqman reverse transcription reagent (Applied Biosystems). For each real-time reaction, 4 μl of cDNA was used combined with primer pairs for mouse Actb, Il6, Hspa5 and Chop. Real time transcription-PCR was performed using Sybr green and an ABI 7900 RT–PCR machine (Applied Biosystems). The fold change in mRNA levels was determined using the comparative threshold cycle (C ) method. Target gene transcription was normalized to the levels of Actb mRNA. Cytokine levels in mouse serum and supernatants of infected BMDMs were measured using either a multiplex cytokine/chemokine assay (Bio-Plex 23-plex mouse cytokine assay; Bio-Rad), or via an enzyme-linked immunosorbent assay (IL-6 ELISA; eBioscience), according to the manufacturer’s instructions. Cytotoxicity was determined by using a LDH release assay in supernatant of BMDMs treated as described above. LDH release assay was performed using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega), following manufacturer’s protocol. The percentage of LDH release was calculated as follows: Percentage of LDH release = 100 × (absorbance reading of treated well − absorbance reading of untreated control)/(absorbance reading of maximum LDH release control − absorbance reading untreated control). The kit-provided lysis buffer was used to achieve complete cell lysis and the supernatant from lysis-buffer-treated cells was used to determine maximum LDH release control. HeLa 229 cells (ATCC CCL-2.1) were cultured in 96-well tissue culture plates at a concentration of 4 × 104 cells per well in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% FBS. HeLa 229 cells were transfected with a total of 125 ng of pCMV-HA-Rip2DN or empty control vector per well. 24 h post-transfection HeLa 229 cells were treated with Dextran to enhance infection efficacy before they were infected with 1.7 × 105 Chlamydia bacteria per well. The plates were centrifuged at 2,000 r.p.m. for 60 min at 37 °C, then incubated for 30 min at 37 °C in 5% CO Supernatant was discarded and replaced with DMEM containing 1 μg ml−1 cyclohexine (Sigma Aldrich) and where indicated, 1 μM KIRA6, 10 μM thapsigargin or 10 μg ml−1 MDP, was added to cultures before incubation at 37 °C in 5% CO for 40 h. For gene expression assays, HeLa 229 cells were suspended in Tri-reagent (Molecular Research Center, Cincinnati) and RNA was isolated. Infection efficiency was confirmed in separate plates by staining Chlamydia-infected HeLa 229 cells with anti-Chlamydia MOMP antibody and counting bacteria under a fluorescent microscope. Four independent assays were performed and the standard error of the mean calculated. BMDMs stimulated where indicated with 10 μM thapsigargin for 24 h were lysed in lysis buffer (4% SDS, 100 mM Tris, 20% glycerol) and 10 μg of protein was analysed by western blot using antibodies raised against rabbit TRAF2 (C192, #4724, Cell Signaling), rabbit HSP90 (E289, #4875, Cell Signaling), mouse SGT1 (ab60728, Abcam) and rabbit α/β-tubulin (#2148, Cell Signaling). For tissue culture experiments, statistical differences were calculated using a paired Student’s t-test. To determine statistical significance in animal experiments, an unpaired Student’s t-test was used. To determine statistical significance of differences in total histopathology scores, a Mann–Whitney U-test was used. A two-tailed P value of <0.05 was considered to be significant.


No statistical methods were used to predetermine sample size. The investigators were not blinded to allocation during experiments and outcome assessment C57BL/6 (CD45.2) mice were purchased from Harlan Laboratories (Rehovot, Israel). B6.SJL (CD45.1) mice were bred in-house. Transgenic Ly6a(Sca-1)-EGFP mice and transgenic ROSA26-eYFP (EndoYFP) reporter mice were purchased from Jackson Laboratories. Transgenic nestin-GFP mice were kindly provided by G. N. Enikolopov (Cold Spring Harbour Laboratory, USA). Transgenic c-Kit-EGFP mice were kindly provided by S. Ottolenghi (University of Milano-Bicocca, Italy). Transgenic VE-cadherin (Cdh5, PAC)-CreERT2 mice were kindly provided by R. H. Adams (Max Planck Institute for Molecular Biomedicine, Germany). Conditional mutants carrying loxP-flanked Cxcr4 were provided by D. Scadden (Harvard University, Cambridge, USA). Conditional mutants carrying loxP-flanked Fgfr1 and Fgfr2 (Fgfr1/Fgfr2lox/lox) mice were provided by S. Werner (Institute of Cell Biology, Switzerland) and by D. Ornitz (Washington University School of Medicine, USA). To induce endothelial-specific Cre activity and gene inactivation/expression, adult VE-cadherin(Cdh5, PAC)-CreERT2 mice interbred with Cxcr4lox/lox (EndoΔCxcr4) or Fgfr1/2lox/lox (EndoΔFgfr1/2) or with ROSA26-eYFP mice (EndoYFP) were injected intraperitoneally (i.p.) with Tamoxifen (Sigma, T5648) at 1 mg per mouse per day for 5 days. Mice were allowed to recover for 4 weeks after tamoxifen injections, before euthanasia and experimental analysis. Mice carrying only VE-cadherin (Cdh5, PAC)-CreERT2 transgene or the Cxcr4lox/lox/Fgfr1/2lox/lox mutations were used as wild-typecontrols to exclude non-specific effects of Cre activation or of floxed alleles mutation. The endothelial Fgfr1/2 deletion was confirmed by qRT–PCR measurements of Cxcr4 and Fgfr1/2 mRNA from isolated BMECs. Male and female mice at 8–12 weeks of age were used for all experiments. All mouse offspring from all strains were routinely genotyped using standard PCR protocols. Sample size was limited by ethical considerations and background experience in stem cell transplantation (bone marrow transplantation) which exists in the laboratory for many years and other published manuscripts in the stem cell field, confirming a significant difference between means. No randomization or blinding was used to allocate experimental groups and no animals were excluded from analysis. All mutated or transgenic mouse strains had a C57BL/6 background. All experiments were done with approval from the Weizmann Institute Animal Care and Use Committee. Mice that were maintained at the Weizmann Institute of Science were bred under defined flora conditions. Two-photon in vivo microscopy procedures that were performed in Harvard Medical School were approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. AMD3100 (Sigma-Aldrich) 5 mg per kg was used to treat mice by subcutaneous (s.c.) injection. Mice were euthanized 30 min later. Recombinant murine FGF-2 (ProSpec) 200 μg per kg was used to treat mice by i.p. injections for seven consecutive days. Neutralizing rat anti-VE-cadherin antibodies or rat IgG (eBioscience) at 50 μg per mouse per day were used to treat mice by intravenous (i.v.) injections for 2 or 5 days. Neutralizing mouse anti-CXCR4 antibodies (12G5 clone) or mouse IgG (eBioscience) at 50 μg per mouse were administered twice, with a 30 min interval, by intravenous (i.v.) injections. To inhibit ROS production, the antioxidant N-acetyl-l-cysteine (NAC; Sigma-Aldrich) was administered by i.p. injection of 130 mg per kg for 2, 5 or 7 days. Mice were euthanized 2–4 h following the final injection. For standard and confocal fluorescent microscopy, femurs were fixed for 2 h in 4% paraformaldehyde, which was replaced and then the samples were washed with 30% sucrose, embedded in optimum cutting temperature compound, and then snap-frozen in N-methylbutane chilled in liquid nitrogen. Sections (5–10 μm) were generated with a CM1850 Cryostat (Leica) at −25 °C with a tungsten carbide blade (Leica) and a CryoJane tape transfer system (Instrumedics), and were mounted on adhesive-coated slides (Leica), fixed in acetone and air-dried. Sections were stained by incubation overnight at 4 °C with primary antibodies, followed by 1 h incubation of secondary antibody at room temperature and in some cases also nuclei labelling by Hoechst 33342 (Molecular Probes) for 5 min at room temperature. Standard analysis (5–6 μm sections) was performed with Olympus BX51 microscope and Olympus DP71 camera. Confocal analysis (10 μm sections) was performed using a Zeiss LSM-710 microscope. In some cases, for BMBV morphological and phenotypical confocal analysis, femurs and tibias were fixed for 2 h in 4% paraformaldehyde, decalcified with 0.5 M EDTA at 4 °C with constant shaking, immersed into 20% sucrose and 2% polyvinylpyrrolidone (PVP) solution for 24 hours, then embedded and frozen in 8% gelatin (porcine) in presence of 20% sucrose and 2% PVP. Sections (80–300 μm) were generated using low-profile blades on a CM3050 cryostat (Leica). Bone sections were air-dried, permeabilized for 10 min in 0.3% Triton X-100, blocked in 5% donkey serum at room temperature for 30 min, and incubated overnight at 4 °C with primary antibodies. Confocal analysis was performed using a Zeiss LSM-780 microscope. Z-stacks of images were processed and 3D-reconstructed with Imaris software (version 7.00, Bitplane). As previously described4, tile scan images were produced by combining the signal of multiple planes along the Z-stalk of bone sections to allow visualization of the distinct types of bone marrow blood vessels and the cells in their surroundings. For the quantifications of blood vessel diameters, a region of 600–700 μm from the growth plate towards the caudal region was selected and diameters for arterial and sinusoidal blood vessels were calculated using ImageJ software on the high-resolution confocal images. Primary and secondary antibodies and relevant information about them are indicated in Supplementary Table 1. For in vivo ROS detection in bone marrow sections, mice were injected i.p. with hydroethidine (Life Technologies) 10 mg per kg, 30 min before euthanasia. For in vivo LDL-uptake detection in bone marrow sections, mice were i.v. injected with Dil-Ac-LDL (BTI) 20 μg per mouse, 4 h before euthanasia. Femurs were immediately collected and processed as mentioned earlier. Bone marrow section analysis for scoring ROShigh cells was performed using ImageJ software (Extended Data Fig. 1). Multiple sections (>16 per mouse) were generated and analysed from at least 4 mice per group of experimental procedure, in order to confirm biological repeats of the observed data. In some cases, images were processed to enhance the contrast in order to allow better evaluation of co-localization of cellular borders and markers. Imaris, Volocity (Perkin Elmer), Photoshop and Illustrator (Adobe) software were used for image processing. For blood vessel imaging in the calvarium of Sca-1-EGFP and nestin-GFP mice, we used a microscope (Ultima Multiphoton; Prairie Technologies) incorporating a pulsed laser (Mai Tai Ti-sapphire; Newport Corp.). A water-immersed 20× (NA 0.95) or 40× (NA 0.8) objective (Olympus) was used. The excitation wavelength was set at 850–910 nm. For intravital imaging, mice were anaesthetized with 100 mg ketamine, 15 mg xylazine and 2.5 mg acepromazine per kg. During imaging, mice were supplied with oxygen and their core temperature was maintained at 37 °C with a warmed plate. The hair on the skullcap was trimmed and further removed using urea-containing lotion and the scalp was incised at the midline. The skull was then exposed and a small steel plate with a cut-through hole was centred on the frontoparietal suture, glued to the skull using cyanoacrylate-based glue and bolted to the warmed plate. To visualize blood vessels, mice were injected i.v. with 2 μl of a 2 μM non-targeted nanoparticles solution (Qtracker 655, Molecular Probes). In some cases, mice were i.v. injected with Dil-Ac-LDL (BTI) 40 μg per mouse, 2 h before their imaging. We typically scanned a 50 μm-thick volume of tissue at 4 μm Z-steps. Movies and figures based on two-photon microscopy were produced using Volocity software (Perkin Elmer). For live imaging of blood vessels permeability and leukocyte bone marrow trafficking, a previously described experimental procedures and a home built laser-scanning multiphoton imaging system29, were used with some modifications. Anaesthesia was slowly induced in mice via inhalation of a mixture of 1.5–2% isoflurane and O . Once induced, the mixture was reduced to 1.35% isoflurane. By making a U-shaped incision on the scalp, calvarial bone was exposed for imaging and 2% methocellulose gel placed on it for refractive index matching. For bone marrow blood vessel permeability studies, mice were positioned in heated skull stabilization mount which allowed access to the eye for on-stage retro-orbital injection of 40–60 μl of 10 mg ml−1 70 kDa rhodamine-dextran (Life Technologies). Nestin-GFP (excited at 840 nm) and confocal reflectance (at 840 nm) signals were used to determine a region of interest within the mouse calvarial bone marrow for measurement of permeability. Rhodamine-dextran was injected and was continuously recorded (30 frames per second) for the first 10 min after injection. After video acquisition, mice were removed from the microscope and euthanasized with CO . In some cases, following dextran clearance, the same mice were used for homing experiments to monitor leukocyte cell trafficking in regions and blood vessels that were defined as less or more permeable. For cell homing studies, mice were injected with 2 × 106 DiD-labelled (Life Technologies) lineage depleted immature haematopoietic progenitor cells (Miltenyi depletion) and with 2 × 106 DiI-labelled (Life Technologies) bone marrow MNC isolated from age matched C57BL/6 mice along with 150 μl of 2 nmol per 100 μl Angiosense 750EX (Perkin Elmer) fluorescent blood pool imaging agent, immediately before mounting the mice on a heated stage of a separate confocal/multiphoton microscope. Intravital images of the mouse bone marrow were collected for up to the first 3 h after injection of the cells. After imaging, the mice were removed from the microscope and euthanized with CO . Permeability, blood flow/shear rates and homing experiments were repeated, n = 3 mice each, measuring multiple blood vessels and events, each mouse regarded as an independent experiment, in order to confirm biological repeats of the observed data. The contrast and brightness settings of the images in the figures were adjusted for display purposes only. For permeability studies, the RGB movies were separated into red (Rhodamine-Dextran), green (nestin-GFP), and blue (reflectance) grayscale image stacks. An image registration algorithm (Normalized Correlation Coefficient, Template Matching) was performed on the red stack using ImageJ (v. 1.47p) to minimize movement artefacts within the image stack. Manual selection of regions of interest (ROI) was performed immediately next to individual vessels within the focus. Permeability of the vessels was calculated using the following equation: P is the permeability of the vessel, V is the volume of the ROI next to the vessel, A is the fractional surface area of the vessel corresponding to the ROI, dI/dt is the intensity of the dye in the ROI as a function of time, I is the intensity of the dye inside the corresponding vessel at the beginning of measurement, and I is the intensity of the dye in the ROI at the beginning of measurement. To calculate dI/dt for a given vessel, the change in intensity was measured within the ROI over time and linearly fit the first ~5–40 s of the data. The slope of this linear fit is dI/dt. The ROI intensity curve is only linear for the first 30–40 s, after which it begins to plateau. For cell homing, the number of stationary cells from the calvarial bone marrow images was counted and categorized into two groups: adherent and extravasated. We categorized both cells within the lumen of the vessel and cells in the process of transmigration in the adherent category. Maximum intensity projections of multiple z-stacks of images were used to count the number of cells in the two categories. When there was a gap between cells and vessels in the two-dimensional projection image, those cells were categorized as extravasated. If any part of a cell overlapped a vessel in the projection image, the corresponding three dimensional z-stack was viewed to determine if the cell had undergone extravasation. When it was unclear if a cell had extravasated, it was always categorized as adherent. For the flow speed measurement, red blood cells (RBCs) were labelled with 15 μM CFSE for 12 min at 37 °C in PBS supplemented with 1 g per litre of glucose and 0.1% BSA. About 0.6 billion RBCs were injected (i.v). 40 μl of rhodamineB-dextran 70 kDa (10 mg ml−1) was retro-orbitally injected immediately before imaging for visualizing bone marrow vasculature. Videos of confocal images of blood vessel (RhodamineB, excitation: 561 nm, emission: 573–613 nm) and labelled RBCs (CFDA-SE, excitation: 491 nm, emission: 509–547 nm) were taken with the speed of 120 frames per second. Individual RBCs were traced over a couple of frames. Total displacement of the RBCs was measured by ImageJ and the speed of blood flow was calculated by: To calculate the shear rate, we assumed that the vessels were straight (straight cylinder) and the blood is an ideal Newtonian fluid with constant viscosity. Under these conditions, the shear rate (du/dr) can be calculated by du/dr = 8×u/d (u is the average blood flow speed which was measured by tracing labelled RBCs and d is the diameter of the blood vessel as measured using ImageJ). Immunostaining signal intensity was analysed with MacsQuant (Miltenyi, Germany) or with a FACS LSRII (BD Biosciences) with FACSDiva software, data were analysed with FlowJo (Tree Star). Data of the expression of molecules by cells was analysed and presented as MFI (mean fluorescent intensity). To acquire single bone marrow cell suspensions, freshly isolated bones were cleaned, flushed and crushed using liver digestion medium (LDM, Invitrogen) supplemented with 0.1% DNaseI (Roche) and further digested for 30 min at 37 °C, under shaking conditions. Following the incubation time, cells were filtered and washed extensively. To isolate and acquire mononuclear cells (MNC) from the peripheral blood PB, blood was collected from the heart using heparinized syringes and MNC were separated using Lymphoprep (Axis-Shield) according to the manufacturer’s instructions. Isolated bone marrow and peripheral blood MNC cells underwent red blood cell lysis (Sigma) before staining. Cells were stained for 30 min at 4 °C in standard flow cytometry buffer with primary antibodies and, where indicated, with secondary antibodies. Information about the primary and secondary antibodies can be found in the antibody information (Supplementary Table 1). For antigens that required intracellular staining, cell surface staining was followed by cell fixation and permeabilization with the Cytofix/Cytoperm kit following the manufacturer’s instructions (BD Biosciences). In case of internal GFP labelled cells, cells were fixed for 20 min with 4% PFA at room temperature, washed and incubated at room temperature for 1 h in 30% sucrose. Cells were washed with flow cytometry buffer and further permeabilized. For intracellular ROS detection, cells were incubated for 10 min at 37 °C with 2 μM hydroethidine (Life Technologies). For glucose uptake detection, cells were incubated for 30 min at 37 °C with the glucose analogue 2-NBDG (Life Technologies). For detection of apoptotic cells, cells were resuspended in annexinV binding buffer (BioLegend) and stained with Pacific Blue AnnexinV (BioLegend). Bone marrow cells were enriched for the lineage negative population, prepared as indicated for flow cytometry and analysed using an ImageStreamX (Amnis) machine. Samples were visualized and analysed for the expression of markers and antigens with IDEAS 4.0 software (Amnis). Single-stained control cells were used to compensate fluorescence between channel images. Cells were gated for single cells with the area and aspect ratio features or, for focused cells, with the Gradient RMS feature. Cells were then gated for the selection of positively stained cells only with their pixel intensity, as set by the cutoff with IgG and secondary antibody control staining. At least 5 samples from 5 mice were analysed to confirm biological repeats of observed data. Detection of mouse calcitonin (Cusabio) and mouse PTH (Cloud-Clone Corp.) levels in bone marrow supernatants was performed according to the manufacturer’s instructions. CFU-GM and CFU-F assays were previously described34. For CFU-Ob assay (also known as mineralized nodule formation assay), CFU-F medium was supplemented with 50 μg ml−1 ascorbic acid and with 10 mM β-glycerophosphate. After 3 weeks, cultures were washed, fixed and stained using Alizarin red for mineralized matrix. The area of mineralized nodules per cultured well was quantified based on image analysis using ImageJ. Bone marrow cells were isolated after sterile bone flushing, crushing and digestion (as previously described). After washing, total bone marrow cells were incubated in medium supplemented with or without 25% blood plasma or supplemented with 20 ng ml−1 TGF-β1 (ProSpec) for 2 h. Plasma was isolated and collected from the upper fraction acquired from the peripheral blood after 5 min centrifugation at 1,500 r.p.m. Bone marrow vascular endothelial barrier function was assessed using the Evans Blue Dye (EBD) assay. Evans Blue (Sigma-Aldrich) 20 mg per kg was injected i.v. 4 h before mice were euthanized. In each experiment, a non-injected mouse was used for control blank measurements. Subsequently, mice were perfused with PBS via the left ventricle to remove intravascular dye. Femurs were removed and formamide was used for bone flushing, crushing and chopping. EBD was extracted in formamide by incubation and shaking of flushed and crushed fractions, overnight at 60 °C. After 30 min centrifugation at 2,000g, EBD in bone marrow supernatants was quantitated by dual-wavelength spectrophotometric analysis at 620 nm and 740 nm. This method corrects the specimen’s absorbance at 620 nm for the absorbance of contaminating haem pigments, using the following formula: corrected absorbance at 620 nm = actual absorbance at 620 nm – (1.426(absorbance at 740) + 0.03). Samples were normalized by subtracting control measurements. Levels of EBD bone marrow penetration per femur were expressed as OD /femur and the fold change in EBD bone marrow penetration was calculated by dividing the controls OD /femur from the treated OD /femur in each experiment. Finally, values were normalized per total protein extract as determined by Bradford assay per sample. For competitive LTR assay, B6.SJL (CD45.1) recipient mice were lethally irradiated (1,000 cGy from a caesium source) and injected 5 h later with 2 × 105 donor-derived (C57BL/6 background, CD45.2) bone marrow cells or with 500 μl of donor-derived whole blood together with 4 × 105 recipient derived (CD45.1) bone marrow cells. Recipient mice were euthanized 24 weeks after transplantation to determine chimaerism levels using flow cytometry analysis. For calculation of competitive repopulating units (CRU), recipient mice were transplanted with limiting dilutions of donor derived bone marrow cells (2.5 × 104 to 2 × 105) together with 2 × 105 recipient derived bone marrow cells. Mice were euthanized after 24 weeks and multi-lineage myelo-lymphoid donor derived contribution in the peripheral blood was assessed using flow cytometry analysis. HSC-CRU frequency and statistical significance was determined using ELDA software (http://bioinf.wehi.edu.au/software/elda/). Lineage negative cells were enriched from total bone marrow cells, taken from c-Kit-EGFP mice, using mouse lineage depletion kit (BD) according to the manufacturer’s instructions. Non-irradiated recipient mice were transplanted by i.v. injection with 2 × 106 c-Kit-EGFP-labelled Lin− cells. Recipient mice were euthanized 4 h after transplantation. Bone marrow cells were isolated from femurs and stained for flow cytometry as described above. Femur cellularity was determined in order to calculate the number of homed CD34−/LSK HSPC per femur. For magnetic isolation of BMECs, freshly recovered bones were processed under sterile conditions as described for BMECs flow cytometry analysis, and post-digestion incubated with biotin rat anti-mouse CD31 antibodies (BD pharmigen) for 30 min at 4 °C. Next, cells were washed and incubated with streptavidin particles plus (BD IMag) for 30 min at 4 °C. Positive selection was performed using BD IMagnet (BD) according to the manufacturer’s instructions (BD Biosciences). BD IMag buffer (BD) was used for washing and for antibodies dilution. Isolated cells were seeded on fibronectin (Sigma-Aldrich) coated wells and cultured overnight in EBM-2 medium (Lonza) supplemented with EGM-2 SingleQuots (Lonza) at 37 °C 5% CO . Non-adhesive cells were removed and adherent cells were collected using accutase (eBioscience). Flow cytometry was applied to confirm endothelial identity and >90% purity of recovered cells. BMEC were further processed to isolate RNA. Total RNA was isolated using TRI-Reagent (Sigma-Aldrich) according to the manufacturer’s protocol. An aliquot of 2 μg of total RNA was reverse-transcribed using Moloney murine leukaemia virus reverse transcriptase (Promega, Madison, WI) and oligo-dT primers (Promega). Quantitative reverse transcribed–polymerase chain reaction (qRT–PCR) was done using the ABI 7000 machine (Applied Biosystems, Foster City, CA) with SYBR Green PCR Master Mix (Applied Biosystems). Comparative quantization of transcripts was assessed relative to hypoxanthine phosphoribosyl transferase (Hprt) levels and amplified with appropriate primers. Primer sequences used were as follows (mouse genes): Cxcr4 forward 5′- ACGGCTGTAGAGCGAGTGTT-3′; reverse 5′- AGGGTTCCTTGTTGGAGTCA-3′; Fgfr1 forward 5′-CAACCGTGTGACCAAAGTGG-3′; reverse 5′-TCCGACAGGTCCTTCTCCG-3′; Fgfr2 forward 5′-ATCCCCCTGCGGAGACA-3′; reverse 5′-GAGGACAGACGCGTTGTTATCC-3′; Hprt forward 5′-GCAGTACAGCCCCAAAATGG-3′; reverse 5′-GGTCCTTTTCACCAGCAAGCT-3′. All statistical analyses were conducted with Prism 4.0c version or Excel (*P < 0.05, **P < 0.01, ***P < 0.005; NS, not significant). All data are expressed as mean ± standard error (s.e.m) and all n numbers represent biological repeats. Unless indicated otherwise in figure legends, a Student’s two-tailed unpaired t-test was used to determine the significance of the difference between means of two groups. One-way ANOVA or two-way ANOVA was used to compare means among three or more independent groups. Bonferroni post-hoc tests were used to compare all pairs of treatment groups when the overall P value was <0.05. A normal distribution of the data was tested using the Kolmogorov–Smirnov test if the sample size allowed. If normal-distribution or equal-variance assumptions were not valid, statistical significance was evaluated using the Mann–Whitney test and the Wilcoxon signed rank test. Animals were randomly assigned to treatment groups. Tested samples were assayed in a blinded fashion.

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