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BSA
Montreal, Canada

The British South Africa Company was established following the amalgamation of Cecil Rhodes' Central Search Association and the London-based Exploring Company Ltd which had originally competed to exploit the expected mineral wealth of Mashonaland but united because of common economic interests and to secure British government backing. The company received a Royal Charter in 1889 modelled on that of the British East India Company. Its first directors included the Duke of Abercorn, Rhodes himself and the South African financier Alfred Beit. Rhodes hoped BSAC would promote colonisation and economic exploitation across much of south-central Africa, as part of the "Scramble for Africa". However, his main focus was south of the Zambezi, in Mashonaland and the coastal areas to its east, from which he believed the Portuguese could be removed by payment or force, and in the Transvaal, which he hoped would return to British control.It has been suggested that Rhodes' ambition was to create a zone of British commercial and political influence from "Cape to Cairo", but this was far beyond the resources of any commercial company to achieve and would not have given investors the financial returns they expected. BSAC was created in the expectation that the gold fields of Mashonaland would provide funds for the development of other areas of Central Africa, including the mineral wealth of Katanga. When the expected wealth of Mashonaland did not materialise and Katanga was acquired by the Congo Free State, the company had little money left after building railways for significant development, particularly in areas north of the Zambezi. BSAC regarded its lands north of the Zambezi as territory to be held as cheaply as possible for future, rather than immediate, exploitation.As part of administering Southern Rhodesia until 1923 and Northern Rhodesia until 1924, BSAC formed what were originally paramilitary forces, but which later included more normal police functions. In addition to the administration of Southern and Northern Rhodesia, BSAC claimed extensive landholdings and mineral rights in both the Rhodesias and, although its land claims in Southern Rhodesia were nullified in 1918, its land rights in Northern Rhodesia and its mineral rights in Southern Rhodesia had to be bought out in 1924 and 1933 respectively, and its mineral rights in Northern Rhodesia lasted until 1964. BSAC also created the Rhodesian railway system and owned the railways there until 1947. Wikipedia.


Argiles J.M.,University of Barcelona | Anguera A.,Rottapharm S.L. | Stemmler B.,BSA
Clinical Nutrition | Year: 2013

Cachexia is a multiorganic syndrome associated with cancer, characterized by body weight loss, muscle and adipose tissue wasting and inflammation, being often associated with anorexia. The aim of the present review is to examine the impact of megestrol acetate in the treatment of cancer cachexia, both at the biochemical and physiological level taking into account new experimental data related to protein muscle metabolism. Based on experimental evidence, it is concluded that megestrol acetate is a good candidate for muscle wasting treatment and future studies addressed at the interaction between the drug and protein turnover in human skeletal muscle should be performed. © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. Source


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K14-CreER transgenic mice23 were kindly provided by E. Fuchs, Rockefeller University; Inv-CreER were generated in our laboratory8. Ptch1fl/fl mice24 and Rosa/SmoM2-YFP mice25 were obtained from the JAX repository. p53fl/fl (ref. 26) mice were obtained from the National Cancer Institute at Frederick. Mouse colonies were maintained in a certified animal facility in accordance with European guidelines. Experiments involving mice presented in this work were approved by Comité d’Ethique du Bien Être Animal (Université Libre de Bruxelles) under protocol number 483N, that states that animals should be euthanized if they present tumours that exceed 1 cm in diameter. The BCCs observed in this study were microscopic and ranged from 1.5 mm to 100 μm in diameter and in none of the experiments performed, the tumours exceeded the limit (1 cm in diameter) described in protocol 483N. Female and male animals have been used for all experiments and equal animal gender ratios have been respected in the majority of the analysis, analysis of the different mutant mice was not blind and sample size was calculated to reach statistical significance. The experiments were not randomized. For clonal induction 3-months-old mice were used. K14-CreER/Rosa-YFP, K14-CreER/Rosa-SmoM2, K14-CreER/SmoM2/p53fl/fl and K14-CreER/Ptch1fl/fl mice received an intraperitoneal injection of 0.1 mg of tamoxifen and Inv-CreER/Rosa-YFP, Inv-CreER/Rosa-SmoM2, Inv-CreER/Rosa-SmoM2/p53fl/fl and Inv-CreER/Ptch1fl/fl received a intraperitoneal injection of 2.5 mg of tamoxifen to achieve similar level of recombination in the different models (Extended Data Fig. 1a). Mice were killed and analysed at different time points following tamoxifen administration. The tail, ventral skin and ear skin were embedded in optimal cutting temperature compound (OCT, Sakura) and cut into 5–8 μm frozen sections using a CM3050S Leica cryostat (Leica Microsystems). Immunostainings were performed on frozen sections. Owing to the fusion of SmoM2 with YFP, SmoM2-expressing cells were detected using anti-GFP antibody. Frozen sections were dried and then fixed with 4% paraformaldehyde/PBS (PFA) for 10 min at room temperature and blocked with blocking buffer for 1 h (PBS, horse serum 5%, BSA 1%, Triton 0.1%). Skin sections were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C, washed with PBS for 3 × 5 min, and then incubated with Hoechst solution and secondary antibodies diluted in blocking buffer for 1 h at room temperature. Finally, sections were washed with PBS for 3 × 5 min at room temperature and mounted in DAKO mounting medium supplemented with 2.5% Dabco (Sigma). Primary antibodies used were the following: anti-GFP (rabbit, 1:1,000, BD, A11122), anti-K14 (Chicken, 1:4,000, Covance, PCK-153P-0100) and anti-B4-integrin (rat, 1:200, BD, 553745). The following secondary antibodies were used: anti-rabbit, anti-rat, anti-chicken, conjugated to AlexaFluor488 (Molecular Probes) and to rhodamine Red-X (JacksonImmunoResearch). Images of immunostaining in sections were acquired using an Axio Imager M1 microscope, an AxioCamMR3 camera and the Axiovision software (Carl Zeiss). Whole mounts of tail epidermis were performed as previously described27 and used to quantify the proportion of surviving clones (Extended Data Fig. 2b) as well as the basal suprabasal and total clone size. Specifically, pieces of tail were incubated for 1 h at 37 °C in EDTA 20 mM in PBS in a rocking plate, then using forceps the dermis and epidermis were separated and the epidermis was fixed for 30 min in PFA 4% in agitation at room temperature and washed 3 times with PBS. For the immunostaining, tail skin pieces were blocked with blocking buffer for 3 h (PBS, horse serum 5%, Triton 0.8%) in a rocking plate at room temperature. After, the skin pieces were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C, the next day they were washed with PBS-Tween 0.2% for 3 × 10 min at room temperature, and then incubated with the secondary antibodies diluted in blocking buffer for 3 h at room temperature, washed 2 × 10 min with PBS-Tween 0.2% and washed for 10 min in PBS. Finally, they were incubated in Hoechst diluted in PBS for 30 min at room temperature in the rocking plate, washed 3 × 10 min in PBS and mounted in DAKO mounting medium supplemented with 2.5% Dabco (Sigma). Primary antibodies used were the following: anti-GFP (rabbit, 1:100, BD, A11122), anti-GFP (goat, 1:800, Abcam, Ab6673), anti-active-caspase3 (rabbit, 1:600, R&D, AF835), anti-β4-integrin (rat, 1:200, BD, 553745) and anti-K31 (guinea pig, 1:200, Progen, GP-hHa1). The following secondary antibodies were used: anti-rabbit, anti-rat, anti-chicken, anti-goat and anti-guinea pig, conjugated to AlexaFluor488 (Molecular Probes), to rhodamine Red-X (JacksonImmunoResearch) and to Cy5 (1:400, Jackson ImmunoResearch). Quantification of the proportion of surviving clones, as well as total and basal clone size was determined by counting the number of SmoM2–YFP and YFP-positive cells. in each clone using whole-mount tail epidermis. The different clones were imaged using Z-stacks using a confocal microscope LSM 780 (Carl Zeiss) and orthogonal views were used to count the number of basal and total number of SmoM2–YFP or YFP-positive cells in each clone, as well as the number of active-caspase3-positive cells in each clone. K31 staining was used to classify the clones according to their location in the scale or interscale regions. To measure the kinetics of cell proliferation, a 24 h continuous pulse of EdU followed with a continuous pulse of BrdU were performed. Specifically, mice received at t = 0 an intraperitoneal injection of EdU (1 mg ml−1) and 0.1 mg ml−1 EdU was added to their drinking water for 24 h. The next days the mice received a daily intraperitoneal injection of BrdU (10 mg ml−1) and 1 mg ml−1 of BrdU was added to their drinking water during the 8 days of the continuous BrdU pulse. Mice were killed at different time points and whole-mount stainings for the tail were performed. The pieces of tail were first stained for GFP (following the protocol described in the previous section). Second, EdU staining was performed following the manufacturer’s instructions (Invitrogen). The pieces of tail were then washed in PBS and fixed again in PFA 4% for 10 min. After they were washed in PBS, incubated for 20 min in HCl 1 M at 37 °C, washed three times with PBS-Tween 0.2% and incubated overnight with Alexa-647-coupled anti-BrdU antibody (mouse, 1:200, BD). The next day the tail pieces were washed in PBS, incubated in Hoechst for 30 min at room temperature in the rocking plate, washed 3 × 10 min in PBS and mounted in DAKO mounting medium supplemented with 2.5% Dabco (Sigma). To quantify the number of cells that incorporated EdU and/or BrdU, Z-stacks were acquired for each individual clone and orthogonal views used to count. For p53 immunohistochemistry, 4-μm paraffin sections were deparaffinized, rehydrated, followed by antigen unmasking performed for 20 min at 98 °C in citrate buffer (pH 6) using the PT module. Endogenous peroxydase was blocked using 3% H O (Merck) in methanol for 10 min at room temperature. Endogenous avidin and biotin were blocked using the Endogenous Blocking kit (Invitrogen) for 20 min at room temperature. In p53 staining, nonspecific antigen blocking was performed using M.O.M. Basic kit reagent. Mouse anti-p53 antibody (clone 1C12; Cell Signaling) was incubated overnight at 4 °C. Anti-mouse biotinylated with M.O.M. Blocking kit, Standard ABC kit, and ImmPACT DAB (Vector Laboratories) was used for the detection of horseradish peroxidase (HRP) activity. Slides were then dehydrated and mounted using SafeMount (Labonord). For the quantification of the clone morphology of SmoM2-expressing clones in the scale and interscale regions (Fig. 1f), we counted in K14-CreER/Rosa-SmoM2 mice, 128, 109, 76, 195, 168 and 142 clones in the interscale region; 141, 116, 74, 94, 78 and 69 clones in the scale region from 3, 4, 4, 6, 4 and 5 independent experiments at 1, 2, 4, 8, 12 and 24 w respectively. In Inv-CreER/Rosa-SmoM2 mice, 104, 78, 42, 127, 160 and 344 clones were counted in the interscale region; 94, 54, 99, 90, 99 and 39 clones in the scale region from 4, 4, 4, 5, 4 and 8 independent experiments at 1, 2, 4, 8, 12 and 24 weeks, respectively. For the analysis of the clone size of the K14-CreER/Rosa-YFP mice (Fig. 2a, c and Extended Data Fig. 2), we counted clones (both in scale and interscale) from two independent experiments at 1 week and 2 weeks, five independent experiments at 4 weeks, three independent experiments at 8 weeks, two independent experiments at 12 weeks and four independent experiments at 24 weeks. For the analysis of the clone size of the Inv-CreER/Rosa-YFP mice (Fig. 2b, c, Extended Data Fig. 2), we counted clones (both in scale and interscale) from two independent experiments at 1 week and 2 weeks, five independent experiments at 4 weeks, three independent experiments at 8 weeks, four independent experiments at 12 weeks and three independent experiments at 24 weeks (see raw data in cited figures (Source Data)). For the clonal persistence of the K14-CreER/Rosa-YFP mice (Fig. 2e and Extended Data Fig. 2), we counted 167, 176, 129, 100, 47 and 246 clones in interscale and 184, 109, 75, 66, 19 and 103 clones in scale from 4, 5, 5, 5, 2 and 4 independent experiments at 1, 2, 4, 8, 12 and 24 w respectively. For 24 weeks, we counted several areas per mice as the number of clones was reduced (see Source Data). For the clonal persistence of the Inv-CreER/Rosa-YFP mice (Fig. 2e and Extended Data Fig. 2), we counted 138, 95, 25, 31, 76 and 54 clones in interscale and 12, 17, 7, 8, 20 and 10 clones in scale from 2, 4, 2, 3, 4 and 3 independent experiments at 1, 2, 4, 8, 12 and 24 weeks respectively. For 12 and 24 weeks, we counted several areas per mice as the number of clones was low (see Source Data). For the analysis of the clone size of the Inv-CreER/Rosa-SmoM2 mice (Fig. 3b, f, h, Extended Data Figs 5, 6), we counted clones (both in scale and interscale) from two independent experiments at 1 week and 2 weeks, from four independent experiments at 4 weeks, from six independent experiments at 8 weeks, from six independent experiments at 12 weeks and from four independent experiments at 24 weeks (see Source Data). For the clonal persistence of the Inv-CreER/Rosa-SmoM2 mice (Extended Data Figs 5, 6), we counted 65, 39, 71, 51, 27, 18 clones in interscale and 67, 27, 47, 31, 12 and 6 clones in scale from 2, 2, 4, 3, 2 and 2 independent experiments at 1, 2, 4, 8, 12 and 24 weeks, respectively (see Source Data). For the analysis of the clone size of the K14-CreER/Rosa-SmoM2 mice (Fig. 4b, f, h and Extended Data Figs 6, 7), we counted clones (both in scale and interscale) from three independent experiments at 1 week, from two independent experiments at 2 weeks, 4 weeks, from six independent experiments at 8 weeks, from four independent experiments at 12 weeks and from two independent experiments at 24 weeks (see Source Data). For the clonal persistence of the K14-CreER/Rosa-SmoM2 mice (Extended Data Figs 6, 7), we counted 122, 63, 81, 79, 74 and 68 clones in interscale and 89, 46, 37, 42, 31 and 16 clones in scale from 4, 3, 4, 4, 4 and 4 independent experiments at 1, 2, 4, 8, 12 and 24 weeks respectively (see Source Data). For the cell proliferation kinetics experiments in the Inv-CreER/Rosa-SmoM2 mice (Fig. 3c, e): at 4 weeks after induction, we counted 33 clones from 3 independent experiments for 2 days of continuous BrdU, 30 clones from 2 independent experiments for 4 days of continuous BrdU, 33 clones from 2 independent experiments for 6 days of continuous BrdU. At 8 weeks after induction, we counted 41 clones from n = 3 mice for 2 days of continuous BrdU, 16 clones from 2 independent experiments for 4 days of continuous BrdU, 30 clones from 2 independent experiments for 6 days of continuous BrdU and 24 clones from 2 independent experiments for 8 days of continuous BrdU. At 12 weeks after induction, we counted 19 clones from 2 independent experiments for 2 days of continuous BrdU, 26 clones from 2 independent experiments for 4 days of continuous BrdU, 27 clones from 2 independent experiments for 6 days of continuous BrdU and 31 clones from 2 independent experiments mice for 8 days of continuous BrdU. For the 2 weeks after induction data point, we use solely continuous BrdU incorporation, and counted 54 clones from two independent experiments. For the cell proliferation kinetics experiments in the K14-CreER/Rosa-SmoM2 mice (Fig. 4c, e): at 4 weeks after induction, we counted 56 clones from 3 independent experiments for 2 days of continuous BrdU, 39 clones from 3 independent experiments for 4 days of continuous BrdU, 29 clones from 3 independent experiments for 6 days of continuous BrdU. At 8 weeks after induction, we counted 30 clones from 2 independent experiments for 2 days of continuous BrdU, 25 clones from 2 independent experiments for 4 days of continuous BrdU, 63 clones from 3 independent experiments for 6 days of continuous BrdU and 41 clones from 3 independent experiments for 8 days of continuous BrdU. At 12 weeks after induction, we counted 20 clones from 2 independent experiments for 2 days of continuous BrdU, 21 clones from 2 independent experiments for 4 days of continuous BrdU, 28 clones from 2 independent experiments for 6 days of continuous BrdU and 26 clones from two independent experiments for 8 days of continuous BrdU. For the quantification of the clone morphology in absence of p53 interscale (Fig. 5c). For K14-CreER/Rosa-SmoM2/p53fl/fl mice 186, 217, 90, 343, 452 and 543 clones from 3, 3, 2, 3, 5 and 5 independent experiments and for Inv-CreER/Rosa-SmoM2/p53fl/fl 95, 98, 199, 271, 263 and 210 clones from 3, 3, 3, 4, 4 and 4 independent experiments were analysed at 1, 2, 4, 8, 12 and 24 weeks respectively. In the quantification in the scale region (Extended Data Fig. 8b) for K14-CreER/Rosa-SmoM2/p53fl/fl 178, 204, 100, 132, 232 and 120 clones were counted from 3, 3, 2, 3, 5 and 5 independent experiments 1, 2, 4, 8, 12 and 24 weeks respectively. For Inv-CreER/Rosa-SmoM2 82, 127, 167, 136, 62 and 153 clones were counted from 2, 3, 3, 4, 4 and 5 independent experiments 1, 2, 4, 8, 12 and 24 weeks respectively. For the analysis of the clone size of K14-CreER/Rosa-SmoM2/p53fl/flmice (Fig. 5d, g, Extended Data Fig 8e, d) we counted clones from the interscale from two independent experiments at 1, 2 and 4 weeks, three independent experiments at 8 weeks, four independent experiments at 12 weeks. For the analysis of the clone size of Inv-CreER/Rosa-SmoM2/p53fl/flmice (Fig. 5d, g and Extended Data Fig 8e, d) we counted clones from the interscale from two independent experiments at 1 and 2 weeks, three independent experiments at 4 and 8 weeks, four independent experiments at 12 weeks. For the cell proliferation kinetics experiments in the Inv-CreER/Rosa-SmoM2/p53fl/fl mice (Fig. 5f) at 12 weeks after induction 34 clones from 3 independent experiments were counted. For the cell proliferation kinetics experiments in the K14-CreER/Rosa-SmoM2/p53fl/fl mice (Fig. 5f) at 12 weeks after induction 44 clones from two independent experiments were counted. For the clonal persistence experiments in Inv-CreER/Rosa-SmoM2/p53fl/fl, 132, 78, 68, 58 and 89 clones from 4, 3, 3, 3 and 5 independent experiments were counted at 1, 2, 4, 8, 12 and 24 weeks and in K14-CreER/Rosa-SmoM2/p53fl/fl mice 124, 82, 53, 76 and 100 clones were counted from 4, 3, 2, 3 and 4 independent experiments at 1, 2, 4, 8 and 12 weeks respectively (Extended Data Fig. 8e) (see Source Data). Source Data includes the persistence and clone size quantifications of K14-CreER/Rosa–YFP, Inv-CreER/Rosa–YFP, K14-CreER/Rosa-SmoM2, Inv-CreER/Rosa-SmoM2, for K14-CreER/Rosa-SmoM2/p53fl/fl and Inv-CreER/Rosa-SmoM2/p53fl/fl animals at different timepoints.


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No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. CENP-LN was produced as a GST fusion construct from insect cells using the MultiBac expression system31. Specifically, a coding sequence expressing 3C cleavable GST-tagged CENP-L was sub-cloned into MCS2, and the coding sequence of CENP-N was sub-cloned into MCS1 of pFL. Bacmid was then produced from EMBacY cells31, and subsequently used to transfect Sf9 cells and produce baculovirus. Baculovirus was amplified through three rounds of amplification and used to infect Tnao38 cells32. Cells infected with the GST- CENP-L/CENP-N virus were cultured for 72 h before harvesting. Cells were washed and resuspended in lysis buffer (50 mM Na-HEPES, 300 mM NaCl, 10% glycerol, 4 mM 2-mercaptoethanol, 1 mM MgCl pH 7.5). Resuspended cells were lysed by sonication in the presence of Benzonase before clearance at 100,000g at 4 °C for 1 h. Cleared lysate was passed over GSH-Sepharose, before extensive washing with lysis buffer. GST-CENP-L/CENP-N complex was then eluted in lysis buffer + 20 mM reduced glutathione. Eluted protein was concentrated in a 30 kDa Amicon-Ultra-15 Centrifugal Filter (Millipore) in the presence of GST-tagged 3C protease. Concentrated protein was then loaded onto a Superdex 200 16/600 column equilibrated in 20 mM Na- HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol. A 5 ml GSH-Sepharose FF column was connected in series after the Superdex 200 column to trap GST, un-cut GST-CENP-L/CENP-N and GST-tagged 3C protease. Peak fractions corresponding to CENP-L/CENP-N were collected and again concentrated in a 30 kDa MWCO concentrator to approximately 50–100 μM before being flash frozen in liquid N and stored at −80 °C. Synthetic, codon-optimized DNA (Geneart), encoding the human CENP-C1–544His, CENP-C189–544, or CENP-C545–943 was sub-cloned into pFL or pFG (containing an N-terminal 3C cleavable GST) vectors, respectively, by restriction cloning with the enzymes BamHI and SalI. A non-cleavable histidine tag comprising six histidines (His6-tag) was introduced C-terminally of CENP-C1–544His, a tobacco etch virus (TEV) cleavage site was introduced N-terminal of CENP-C545–943. Tnao38 cells expressing CENP-C1–544His, CENP-C189–544, or CENP-C545–943 were resuspended in lysis buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 2 mM β-mercaptoethanol) and lysed by sonication before centrifugation at 100,000g at 4 °C for 1 h. The cleared lysates were incubated with Ni-NTA Agarose beads (for CENP-C1–544His), GST-Trap affinity column (GE Healthcare, for CENP-C189–544) or Glutathione Sepharose 4 Fast Flow beads (for CENP-C545–943) at 4 °C for 2 h. After washing with 70 column volumes of lysis buffer, CENP-C1–544His was eluted with lysis buffer supplemented with 200 mM Imidazole, CENP-C189–544 was eluted in lysis buffer supplemented with 30 mM reduced glutathione, and CENP-C545–943 was cleaved off the beads in 16 h at 4 °C by addition of TEV protease. After elution, proteins were diluted in buffer A (20 mM HEPES pH 7.5, 5% glycerol, 1 mM TCEP, to achieve a final concentration of 300 mM NaCl), loaded onto a pre-equilibrated HiTrap Heparin HP column, and eluted with a linear gradient of buffer B (20 mM HEPES pH 7.5, 2 M NaCl, 5% glycerol, 1 mM TCEP) in a gradient from 300 to 1200 mM NaCl. Fractions containing CENP-C1–544His and CENP-C545–943 were loaded onto a Superdex 200 16/60 SEC column pre-equilibrated in SEC buffer (10 mM HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol, 2 mM TCEP). For CENP-C189–544, the GST tag was cleaved using 3C protease and the protein concentrated in a 10 kDa MWCO concentrator. The protein was then further purified by SEC as described for the other two constructs. SEC fractions containing CENP-C1–544His, CENP-C189–544, or CENP-C545–943 were concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C. NDC80-GFP complexes were constructed with a C-terminal fusion of GFP to HEC1. The unlabelled NDC80 complex was constructed with an N-terminal fusion of a His6-tag to SPC25. Construct for insect cell expression exploited the MultiBac baculovirus expression system31. Bacmid was then produced from EMBacY cells, and subsequently used to transfect Sf9 cells and produce baculovirus. Baculovirus was amplified through three rounds of amplification and used to infect Tnao38 cells. Cells infected with virus expressing untagged NDC80 were cultured for 72 h before harvesting. Cells were washed and resuspended in lysis buffer (25 mM Na-HEPES, 300 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM MgCl pH 7.5 and 1 mM PMSF). Resuspended cells were lysed by sonication in the presence of Benzonase before clearance at 100,000g at 4 °C for 1 h. Cleared lysate was passed over Ni-Sepharose, before extensive washing with lysis buffer. The Ndc80 complex was then eluted in lysis buffer + 250 mM imidazole. Eluted protein was diluted to 50 mM NaCl using buffer A (25 mM Na-HEPES, 10% glycerol, 1 mM TCEP) and loaded on a ResQ anion-exchange column. The NDC80-GFP was eluted using a salt gradient over 30 column volumes to 500 mM NaCl using buffer B (25 mM Na-HEPES, 1,000 mM NaCl, 10% glycerol, 1 mM TCEP). The eluted protein was concentrated in a 30-kDa Amicon-Ultra-15 Centrifugal Filter (Millipore) and the concentrated protein was then loaded onto a Superdex 200 16/600 column equilibrated in 10 mM Na- HEPES pH 7.5, 150 mM NaCl, 2.5% glycerol, pH 7.5. Peak fractions containing the NDC80 complex were collected and again concentrated in a 30 kDa MWCO concentrator to approximately 10 μM before being flash frozen in liquid N and storage at −80 °C. Codon-optimized human CENP-I57–756 (57-C) was subcloned in a MultiBac pFL-derived vector31 with an N-terminal TEV cleavable His6-tag, under the control of the polh promoter. A complementary DNA (cDNA) segment encoding human CENP-M isoform 1 was subcloned in the second MCS of the same vector, under the control of the p10 promoter. Simultaneously, a second pFL-based vector was created with untagged CENP-H and CENP-K under the control of the polh and p10 promoters, respectively. The CENP-I/M vector was then linearized with BstZ171, and the expression region of the CENP-H/K vector was PCR amplified with primers designed for sequence and ligation independent cloning (SLIC) of the PCR fragment into the linearized CENP-I/M vector. The SLIC reaction was then performed to produce a single pFL-based vector with four expression cassettes. Constructs were sequence verified. Baculovirus was then produced and amplified with three rounds of amplification. Expression of CENP-HI57-CKM complex was performed in TnAo38 cells, using a virus:culture ratio of 1:40. Infected cells were incubated for 72 h at 27 °C. Cell pellets were harvested, washed in 1× PBS, and finally resuspended in a buffer containing 50 mM HEPES 7.5, 300 mM NaCl, 1 mM MgCl , 10% glycerol, 5 mM imidazole, 2 mM β-mercaptoethanol, 0.1 mM AEBSF, and 2.5 units per millitre Benzonase (EMD/Millipore). Cells were lysed by sonication, and cleared for 1 h at 100,000g. Cleared cell lysate was then run over a 5 ml Talon superflow column (Clontech) and then washed with 50 mM HEPES 7.5, 1 M NaCl, 10% glycerol, 5 mM imidazole, and 2 mM β-mercaptoethanol. CENP-HI57-CKM complex was eluted with a gradient of 5–300 mM imidazole, and the fractions containing CENP-HI57-CKM pooled, and the His tag cleaved overnight at 4 °C. CENP-HI57-CKM in solution was then adjusted to a salt concentration of 100 mM and a pH of 6.5, before loading on a 6 ml Resource S ion-exchange column (GE Healthcare), equilibrated in 20 mM MES 6.5, 100 mM NaCl, 2 mM β-mercaptoethanol. CENP-HI57-CKM was then eluted with a gradient of 100–1,000 mM NaCl over 20 column volumes, and peak fractions corresponding to CENP-HI57-CKM were pooled and concentrated in a 50 kDa MW Amicon concentrator (Millipore). CENP-HI57-CKM was then loaded onto a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES 7.5, 150 mM NaCl, 2.5% glycerol, 2 mM TCEP. The sample was concentrated and flash frozen in liquid N before use. CENP-HI57-CKM complex was labelled using the Alexa Fluor 405 C5 Maleimide kit (Thermo Fisher Scientific). A cDNA segment encoding residues 459–561 (the histone fold, HF) of human CENP-T isoform 1, was subcloned in pGEX-6P-2rbs vector as a C-terminal fusion to GST, with an intervening 3C protease site. A cDNA segment encoding human CENP-W was subcloned in the second cassette of the same vector. Similarly, a synthetic cDNA segment encoding human CENP-X isoform 1, codon-optimized for expression in bacteria, was subcloned in pGEX-6P-2rbs vector as a C-terminal fusion to GST, with an intervening 3C protease site. Also, a cDNA segment encoding human CENP-S isoform 1, was subcloned in the second cassette of the same vector. Constructs were sequence-verified. The expression and purification procedure was the same for CENP-T/CENP-W and CENP-S/CENP-X complexes. Escherichia coli BL21 Rosetta cells harbouring vectors expressing GST-CENP-T/CENP-W or GST-CENP-X/CENP-S were grown in Terrific Broth at 37 °C to an absorbance at 600 nm (A ) of 0.6–0.8, then 0.3 mM IPTG was added and the culture was grown at 20 °C overnight. Cell pellets were resuspended in lysis buffer (25 mM Tris/HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT) supplemented with protease inhibitor cocktail (Serva), lysed by sonication, and cleared by centrifugation at 48,000g at 4 °C for 1 h. The cleared lysate was applied to Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) pre-equilibrated in lysis buffer, incubated at 4 °C for 2 h, washed with 70 volumes of lysis buffer and subjected to an overnight cleavage reaction with 3C protease. A heparin column (GE Healthcare) was pre-equilibrated in a mixture of 85% buffer A (20 mM Tris/HCl pH 7.5, 5% glycerol, 1 mM DTT) and 15% buffer B (20 mM Tris/HCl pH 7.5, 2 M NaCl, 5% glycerol, 1 mM DTT). The eluate from glutathione beads was directly loaded onto the heparin column and eluted with a linear gradient of buffer B from 300 to 1,200 mM NaCl in ten bed column volumes. Fractions containing CENP-T(HF)/CENP-W or CENP-S/CENP-X were concentrated in 10-kDa-cut-off Vivaspin concentrators (Sartorius) and loaded onto a Superdex 75 size-exclusion chromatography (SEC) column (GE Healthcare) pre-equilibrated in SEC buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM TCEP). SEC was performed under isocratic conditions at a flow rate of 0.5 ml/min. Fractions containing CENP-T(HF)/CENP-W or CENP-S/CENP-X were concentrated. To form the T(HF)WSX complex, T(HF)W was added to SX at a 1.5 molar excess, incubated for 1 h on ice, and then subjected to separation on a Superdex 200 size-exclusion column to separate tetrameric T(HF)SX complex from T(HF)W dimers. Fractions containing the tetrameric T(HF)WSX complex were then concentrated in a 10-kDa MWCO concentrator to a concentration of 50–250 μM, and flash-frozen. Plasmids for the production of X. laevis H2A, H2B, H3 and H4 histones were a gift from D. Rhodes. X. laevis histone expression and purification, refolding of histone octamers or H2A:H2B dimers, and reconstitution of H3 containing mononucleosomes were performed precisely as described33. Plasmids for the production of the ‘601’ 145-bp DNA were a gift from C. A. Davey. DNA production was performed as described33 with no modifications. For Alexa-647-labelled nucleosomes, the 145-bp DNA fragments (601-Widom) were amplified using fluorescently labelled primers (Sigma-Aldrich). Biotinylated nucleosomes were reconstituted using commercial synthetic 145-bp DNA fragments (601-Widom) (Epicypher). Plasmids for the production of human CENP-A:H4 histone tetramer were a gift of A. F. Straight. Preparations of CENP-A-containing NCPs were performed precisely as described34. For Alexa-647-labelled nucleosomes, the 145-bp DNA fragments (601-Widom) were amplified using fluorescently labelled primers (Sigma-Aldrich, St. Louis, Missouri, USA). Biotinylated nucleosomes were reconstituted using commercial synthetic 145-bp DNA fragments (601-Widom) (Epicypher, Durham, North Carolina, USA). Polycistronic-coexpression plasmid pETDuet–6HisH3.1/CENP-A–H4–6His-H2A–H2B-BFP was generated on the basis of the strategy described previously35 with human histone sequences. The coding sequences of the open reading frames of 6His-H3.1(Ala2–Ile75)/CENP-A(Cys75–Gly140), H4, 6His-H2A1B, and H2B1J-TagBFP were sub-cloned between NcoI and XhoI sites of pETDuet-1 using conventional cloning techniques and the Gibson cloning36. The H3 and CENP-A segments of the chimaera paste within the α1-helix in a structurally seamless manner. One ribosome-binding site was placed upstream of each open reading frame of these four recombinant histones. A TEV protease site was placed between 6His-tag and H3.1/CENP-A-chimaera and a PreScission protease site was placed between 6His-tag and H2A1B to allow tag-removal during protein purification. Protein expression and purification of BFP-labelled H3.1/CENP-A-chimaera histone octamer followed a previous study35 with minor modifications. Purification of the octamer was done according to the previous study35 with minor modifications. After Ni-affinity purification, the octamers were incubated for 15 h at 4 °C with His-TEV protease and His-PreScission protease in buffer A containing 20 mM Tris-HCl pH 8.0, 1.0 M sodium chloride, 1 mM tris(2-carboxyethyl)phosphine (TCEP). The tag-removed octamers were concentrated in buffer B (20 mM Tris-HCl pH 8.0, 2.0 M sodium chloride, 1 mM TCEP) and further purified using Superdex 200 10/300 GL gel-filtration column (GE Healthcare) equilibrated with buffer B. Fractions containing the octamers were pooled, concentrated and stored at −80 °C until used for nucleosome reconstitution. Analytical SEC was performed on a custom-made Superose 6 5/200 in a buffer containing 20 mM HEPES, 300 mM NaCl, 2.5% glycerol, 2 mM TCEP, pH 7.5 on an ÄKTAmicro system. As indicated, the following additional columns were used: Superdex 200 5/150 Increase and Superose 6 5/150. All samples were eluted under isocratic conditions at 4 °C in SEC buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol, 2 mM TCEP) at a flow rate of 0.2 ml/min. Elution of proteins was monitored at 280 nm. Fractions (100 μl) were collected and analysed by SDS–PAGE and Coomassie blue staining. To detect the formation of a complex, proteins were mixed at the indicated concentrations in 50 μl, incubated for at least 2 h on ice and then subjected to SEC. Coverslips and glass slides were cleaned by sonication in isopropanol and 1 M KOH or 1% Hellmanex and 70% ethanol, respectively. After functionalization of coverslips with 5% biotinylated poly-l-lysine- PEG for 30 min, flow cells were created with a volume of 10–15 μl. Flow cells were passivated with 1% pluronic F-127 for 1 h and coated with avidin for 30–45 min. After incubation with 10 nM microtubules (10% biotinylated, 10% rhodamine labelled, Cytoskeleton, polymerized according to the manufacturer’s instructions) for 10–20 min, proteins (400 nM) were added in 80 mM Pipes (pH6.8), 125 mM KCl, 1 mM EGTA, 1 mM MgCl and 20 μM Taxol). Flow cells were sealed with wax and imaged with spinning disk confocal microscopy on a 3i Marianas system (Intelligent Imaging Innovations, Göttingen, Germany) equipped with Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany), a CSU-X1 confocal scanner unit (Yokogawa Electric Corporation, Tokyo, Japan), Plan-Apochromat 100×/1.4 numerical aperture DIC oil objective (Zeiss), Orca Flash 4.0 sCMOS Camera (Hamamatsu, Hamamatsu City, Japan) and controlled by Slidebook Software 6.0 (Intelligent Imaging Innovations). Images were acquired as z-sections at 0.27 μm and maximal intensity projections were made with Slidebook Software 6.0 (Intelligent Imaging Innovations). GST pulldown experiments were performed using pre-blocked GSH Sepharose beads in pulldown buffer (10 mM HEPES pH 7.5, 200 mM NaCl, 0.05% Triton, 2.5% glycerol, 1 mM TCEP). GST-CENP-LN as bait at a 1 μM concentration was incubated with NCPs as prey at a 3 μM concentration. The bait was loaded to 12 μl preblocked beads, before the prey was added. At the same time, 1 μg of each protein was added into Laemmli sample loading buffer for the input gel. The reaction volume was topped up to 40 μl with buffer and incubated at 4 °C for 1 h under gentle rotation. Beads were spun down at 500g for 3 min. The supernatant was removed and beads washed twice with 250 μl buffer. Supernatant was removed completely, samples boiled in 15 μl Laemmli sample loading buffer and run on a 14% SDS–PAGE gel. Bands were visualized with Coomassie brilliant blue staining. Preblocking of GSH sepharose beads 750 μl of GSH Sepharose beads were washed twice with 1 ml washing buffer (20 mM HEPES pH 7.5, 200 mM NaCl) and incubated in 1 ml blocking buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 500 μg/μl BSA) overnight at 4 °C rotating. Beads were washed five times with 1 ml washing buffer and resuspended in 500 μl washing buffer to have a 50/50 slurry of beads and buffer. For CENP-C silencing, we used a single siRNA (target sequence: 5′-GGAUCAUCUCAGAAUAGAA-3′; obtained from Sigma-Aldrich), targeting the coding region of endogenous CENP-C mRNA. For an efficient depletion, siRNA for CENP-C was transfected at a concentration of 60 nM for 72 h. For CENP-M silencing, we used a combination of three siRNA duplexes (target sequences: 5′-ACAAAAGGUCUGUGGCUAA-3′; 5′-UUAAGCAGCUGGCGUGUUA-3′; 5′-GUGCUGACUCCAUAAACAU-3′; purchased from Thermo Scientific, Carlsbad, California, USA) targeting the 3′-UTR of endogenous CENP-M. CENP-M siRNA duplexes were used at 20 nM each for 72 h as published3. For CENP-H a single siRNA (target sequence: 5′-CUAGUGUCUCAUGGAUAA-3′ obtained from Dharmacon) targeting the coding region of endogenous CENP-H mRNA was used at 100 nM for 72 h. For CENP-L a single siRNA (target sequence: 5′-UUUAUCAGCCACAAGAUUA-3′ obtained from Dharmacon) targeting the coding region of endogenous CENP-L was used at 100 nM for 72 h. Transfections of RNAi were performed with HyPerFect (QIAGEN) according to the manufacturer’s instructions. Phenotypes were analysed 96 h after first siRNA addition and protein depletion was monitored by western blotting or immunofluorescence. Constructs were created by cDNA subcloning in pcDNA5/FRT/TO-mCherry-IRES vector, a modified version of pcDNA5/FRT/TO vector (Invitrogen). pcDNA5/FRT/TO vector (Invitrogen) is a tetracycline-inducible expression vector designed for use with the Flp-In T-REx system. It carries a hybrid human cytomegalovirus/TetO2 promoter for high-level, tetracycline-regulated expression of the target gene. Parental Flp-In T-REx HeLa cells used to generate stable doxycycline-inducible cell lines were a gift from S. Taylor (University of Manchester, Manchester, UK). They were grown at 37 °C in the presence of 5% CO in Dulbecco’s Modified Eagle’s Medium (DMEM; PAN Biotech) supplemented with 10% TET-free Fetal Bovine Serum (Invitrogen) and 2 mM l-glutamine (PAN- Biotech, 250 μg/ml hygromycin (Invitrogen, Carlsbad, California, USA) and 4 μg/ml blastidicin (Invitrogen, Carlsbad, California, USA). The cell line was regularly tested for mycoplasma contamination. RNAi-depleted cells for various CCAN components were harvested by trypsinization and lysed by incubation in lysis buffer (75 mM HEPES pH 7.5, 150 mM KCl, 1.5 mM EGTA, 1.5 mM MgCl , 10% glycerol, 0.075% NP-40, 90 U/ml benzonase (Sigma)), protease inhibitor cocktail (Serva) at 4 °C for 15 min followed by sonication and centrifugation. Cleared lysate was washed with lysis buffer, resuspended in Laemmli sample buffer, boiled, and analysed by western blotting using 12% NuPAGE gels (Life Technologies). The following antibodies were used: anti-Vinculin (mouse monoclonal, clone hVIN-1; 1:15,000; Sigma-Aldrich, V9131), anti-α-tubulin (mouse monoclonal, Sigma-Aldrich T9026), anti-CENP-C (rabbit polyclonal antibody SI410 raised against residues 23-410 of human CENP-C; 1:1,200; ref. 10), anti-CENP-HK (rabbit polyclonal antibody SI0930 raised against the full length human CENP-HK complex; 1:1,000), anti-CENP-M (rabbit polyclonal antibody raised against the full length human CENP-M), anti-CENP-L (rabbit polyclonal, Acries antibodies 17007-1-AP). Secondary antibodies were affinity-purified anti-mouse (Amersham, part of GE Healthcare), anti-rabbit or anti-mouse (Amersham) conjugated to horseradish peroxidase (1:10,000). After incubation with ECL western blotting system (GE Healthcare), images were acquired with ChemiDocTM MP System (BioRad). Levels were adjusted with ImageJ and Photoshop and images were cropped accordingly. Flp-In T-REx HeLa cells were grown on coverslips pre-coated with 0.01% poly-l-lysine (Sigma). Cells were fixed with PBS/PHEM- paraformaldehyde 4% followed by permeabilization with PBS/PHEM-Triton 0.5%. The following antibodies were used for immunostaining: CREST/anti-centromere antibodies (human auto-immune serum, 1:100; Antibodies, Davis, California), anti-CENP-C (SI410; 1:1,000, or the directly Alexa488 conjugated form of this antibody 1:400), anti-CENP-A mouse monoclonal (Gene Tex GTX13939, 1:500) anti-CENP-HK (SI0930; 1:800 or the Alexa488 directly conjugated form of this antibody 1:800). Rodamine Red-conjugated, DyLight405-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA. Alexa Fluor 647-labelled secondary antibodies were from Invitrogen. Coverslips were mounted with Mowiol mounting media (Calbiochem). All experiments were imaged under identical conditions at room temperature using the spinning disk confocal microscopy of a 3i Marianas system (Intelligent Imaging Innovations, Denver, Colorado, USA) equipped with an Axio Observer Z1 microscope (Zeiss, Oberkochen, Germany), a CSU-X1 confocal scanner unit (Yokogawa Electric Corporation, Tokyo, Japan), Plan-Apochromat 63× or 100×/1.4 numerical aperture objectives (Zeiss) and Orca Flash 4.0 sCMOS Camera (Hamamatsu, Hamamatsu City, Japan) and converted into maximal intensity projections TIFF files for illustrative purposes. Quantification of kinetochore signals was performed on unmodified Z-series images using Imaris 7.3.4 software (Bitplane, Zurich, Switzerland). Z-stacks of single cells were processed in Imaris by creating an ellipsoid of 0.3 μm width and 1 μm height, which was positioned on the CREST signal to cover most of the kinetochore signal in all channels. Four background points with equal ellipsoid size and shape were set between kinetochore dots. Intensity values of single kinetochores were exported in a Microsoft Excel file and the average of the background values was subtracted from every kinetochore value. The mean of all kinetochore signals was taken. For each signal, the mean of the corrected values in mock-depleted cells was set to 1. All other values in perturbation experiments were then normalized to this value to derive the fraction of signal for each measured kinetochore protein compared with control cells. Cross-linking analysis of CENP- ANCP:CHIKLMN:KMN complex or CENP-ANCP:CHIKMNL complex was performed with an equimolar mixture of light and heavy-labelled (deuterated) bis[sulfosuccinimidyl] suberate (BS3-d0/d12, Creative Molecules). The complex was incubated with 0.8 mM BS3 for 30 min at 30 °C and the crosslinking reaction was quenched by adding ammonium bicarbonate to a final concentration of 100 mM. Digestion with lysyl enodpeptidase (Wako) was performed at 35 °C, 6 M urea for 2 h (at enzyme–substrate ratio of 1:50 w/w) and was followed by a second digestion with trypsin (Promega) at 35 °C overnight (also at 1:50 ratio w/w). Digestion was stopped by the addition of 1% (v/v) trifluoroacetic acid (TFA). Cross-linked peptides were enriched on a Superdex Peptide PC 3.2/30 column (300 × 3.2 mm) at a flow rate of 25 μl min−1 and water/acetonitrile/TFA, 75:25:0.1 as a mobile phase. Fractions were analysed by liquid chromatography coupled to tandem mass spectrometry using a hybrid LTQ Orbitrap Elite (Thermo Scientific) instrument. Cross-linked peptides were identified using xQuest11. False discovery rates (FDRs) were estimated by using xProphet12 and results were filtered according to the following parameters: FDR = 0.05, min delta score = 0.85, MS1 tolerance window of −4 to 4 ppm, ld-score >22. The crosslinks were visualized using the webserver xVis ( http://xvis.genzentrum.lmu.de/) (ref. 37). EMSA were performed using either Alexa-647-labelled NCPs, or unlabelled NCPs, at 10 nM. Proteins or protein complexes were added to the nucleosomes at the concentrations indicated and incubated in buffer containing 10 mM HEPES, 150 mM NaCl, 2 mM TCEP, 1% glycerol, 1% Ficoll, 2 mg/ml BSA in 10 μL volume. Samples were then run on 0.75% agarose gel in 0.5× TBE at 4 °C. Gels of unlabelled nucleosomes were stained with SYBRGold (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Gels were imaged using a TyphoonTrio scanner (GE Healthcare, Chicago, Illinois, USA). Quantification was performed using ImageJ, and analysis using Prism (Graphpad, La Jolla, California, USA). CENP-A binding data were fitted with a quadratic binding equation. For CENP-A binding by CHIKMLN, a Hill equation with Hill coefficient of 2.07 was applied, without changes in the apparent K . Sedimentation velocity experiments were performed in an Optima XL-A analytical ultracentrifuge (Beckman Coulter, Palo Alto, California, USA) with Epon charcoal-filled double-sector quartz cells and an An-60 Ti rotor (Beckman Coulter, Palo Alto, California, USA). Samples were centrifuged at 203,000g at 10 °C or 20 °C and 500 radial absorbance scans at either 280 nm or at 497 nm (for samples containing CENP-HI57-CKM complex labelled with Alexa Fluor 488) and collected with a time interval of 1 min. Data were analysed using the SEDFIT software38 in terms of continuous distribution function of sedimentation coefficients (c(s)). The protein partial specific volume was estimated from the amino-acid sequence using the program SEDNTERP. Data were plotted using the program GUSSI in the SEDFIT software38. The GUSSI software is also freely available from http://biophysics.swmed.edu/MBR/software.html. Analysis of NCPs or NCPs bound to CENP-LN was performed at 20 °C in 20 mM HEPES pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM EDTA and 2 mM TCEP (leading to values of buffer density of 1.03503 g/ml and viscosity of 1.002 cP). All other experiments were performed at 10 °C in 10 mM HEPES pH 7.5, 2.5% glycerol and 0.3 M NaCl (leading to values of buffer density 1.02001 g/ml and viscosity of 1.307 cP). To calculate the value of the partial specific volume (V‾, inverse of density) for nucleosomes, we took the value of the 0.55 ml/g for the DNA. This gave a value of V‾ = 0.6565 ml/g for the nucleosomes at 20 °C (or 0.65423 ml/g at 10 °C). The value of the partial specific volume for the CENP-LN bound to CENP-A NCPs is 0.692 ml/g at 20 °C (assuming 2:1 stoichiometry). The value for the CHIKMLN and CENP-A NCPs is 0.71666 ml/g at 10 °C (assuming 2:1 stoichiometry). The value for the HIKM is 0.7394 ml/g at 10 °C and the value for CHIKMLN is 0.73380 ml/g at 10 °C. Biotinylated NCPs (0.5 μM) were incubated with prey proteins (1.5 μM or as indicated) for 30 min on ice in a buffer containing 20 mM HEPES, 200 mM NaCl, 0.05% Triton-X100, 2.5% glycerol, 2 mM TCEP in a reaction volume of 40 μl. Ten microlitres of the protein mix were taken as an input. Ten microlitres of pre-equilibrated streptavidin beads (GE Healthcare, Chicago, IL, USA) were then added to the samples and incubated for 2 min. The samples were then spun down, the supernatant removed, and the beads washed once. Laemmli buffer (1×) was then added to the beads, and heated to 95 °C for 1 min to release all the streptavidin from the beads.


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C57BL/6J mice (CD45.2) and B6.SJL-PtprcaPep3b/BoyJ (CD45.1) were purchased from The Jackson Laboratory (Bar Harbour, ME). Prdm16Gt(OST67423)Lex knockout mice31 were obtained from Lexicon Genetics. Conditional Mito-Dendra2 transgenic (Pham) mice32 (B6;129S-Gt(ROSA)26Sortm1(CAG-COX8A/Dendra2)Dcc/J) and E2A-Cre mice33 (B6.FVB-Tg(EIIa-cre)C5379Lmgd/J) were purchased from Jackson Laboratory. Pham mice contain a mitochondrially targeted Dendra preceded by a stoplox sequence in the Rosa locus. These mice were crossed with E2A-Cre mice to effect ubiquitous induction of the MitoDendra2 reporter. Conditional Mfn2 knockout mice34 (B6/129SF1Mfn2tm3Dcc/Mmucd) were obtained from MMRRC and crossed to Vav-Cre transgenic mice35 (B6.Cg-Tg(Vav1-Cre)A2Kio/J) to obtain a homozygous floxed allele Mfn2 allele which generated a B6.Cg-Tg(Vav1-Cre)A2Kio/J;B6/126SF1 Mfn2tm3Dcc/Mmucd mixed mouse strain. All mouse strains were rederived by in vitro fertilization at the Jackson Laboratory. Animals were housed in a specific pathogen-free facility. Experiments and animal care were performed in accordance with the Columbia University Institutional Animal Care and Use Committee. All mice were used at age 8–12 weeks, except in experiments that involved fetal liver cells, when E14.4 embryos were used. Both sexes were used for experiments. Results were analysed in non-blinded fashion. In all experiments, randomly chosen wild type and littermates were used. MEFs were established from approximately 14.5 days post coitum embryos as previously described36 from Prdm16+/− breeder pairs. Briefly, dissected embryo trunks were minced into 1–2 mm fragments, resuspended in 3 ml 0.25% trypsin/EDTA (Gibco, Carlsbad, CA) and passed 20–30 times through a 16 gauge needle. Cell suspensions were incubated at 37 °C for 1 h with frequent agitation. Erythrocytes were lysed with ACK buffer, washed and cells were plated for 3 h in 10% FBS/DMEM. Cells remaining in suspension were aspirated and adherent cells were cultured with fresh media. MEFs were passaged 1:3 every 3 days and cells between passage 2 and 5 were used for all experiments. 293 cells and NIH-3T3 cells were purchased from ATCC (Manassas, VA) and sub-cultured in 10% FBS/DMEM or 10% calf serum/DMEM, respectively. WT and Mfn2−/− MEFs were a kind gift from E. Schon (Columbia University). All lines are tested yearly for mycoplasma contamination and found negative. Prdm16 constructs were generated by subcloning the murine full length (flPrdm16) or truncated (sPrdm16) cDNA into the XhoI/EcoRI sites of the pMSCV-IRES-GFP retroviral expression plasmid. The Mito-dsRed construct was purchased from Addgene (Cambridge, MA) (plasmid 11151). Mfn2 constructs were generated by subcloning the murine Mfn2 cDNA into the EcoRI/BamHI sites of the pLVX-EF1α-IRES-GFP or pLVX-EF1α-IRES-mCherry lentiviral expression plasmid (Clontech). The pGreenFire-Nfat and pGreenFire-CMV gene reporter constructs were purchased from System Biosciences (San Jose, CA) and contained three canonical Nfat response elements (5′- -3′) driving the expression of copGFP and luciferase reporters. The DNDrp1-pcDNA3.1 construct was purchased from Addgene (#45161) and subcloned using the BamHI/EcoRI restriction sites into the pLVX-IRES-GFP vector. Lentiviral 2nd generation packaging construct ΔR8.2 (8455) and pDM2.6 (12259) were purchased from Addgene. The −950/+22 murine MFN2 promoter was constructed by PCR amplification of the RP23-458J18 BAC clone (CHORI, Oakland, CA) and subcloned into the pGL4 luciferase reporter vector (Promega, Madison, WI). All cloning was carried out using KOD hot-start polymerase (Novagen, Billerica, MA) and subcloned for screening and sequencing into the pCR2.1 shuttle vector (Invitrogen, Carlsbad, CA). For peripheral blood analyses, erythrocytes were lysed twice with ACK lysis buffer and nucleated cells were stained with antibody cocktail (Supplementary Table 1) in FACS buffer for 15 min on ice, washed and analysed on a BD FACSCantoII flow cytometer (Becton Dickinson, Mountain View, CA). For bone marrow analyses, cells were isolated using the crushing method and erythrocytes were lysed with ACK lysis buffer followed by 40 μm filtration. bone marrow cells were stained with antibody cocktail in FACS buffer for 30 min on ice, washed and analysed on a BD LSRII flow cytometer (Becton Dickinson, Mountain View, CA). Dead cells were excluded from analyses by gating out 7AAD-positive cells. To isolate purified haematopoietic populations, bone marrow cells were isolated, stained and sorted using a BD Influx cell sorter (Becton Dickinson, Mountain View, CA) into complete media. Data were analysed using FlowJo9.6 (TreeStar Inc., Ashland, OR). Mfn2fl/fl-Vav-Cre fetal liver cells, bone marrow cells or purified LT-HSCs (Lin−cKit+Sca1+CD48−Flt3−CD150+) were transplanted into lethally irradiated (two doses of 478 cGy over 3 h using a Rad Source RS-2000 X-ray irradiator (Brentwood, TN)) recipients together with 2 × 105 competitor cells. As Mfn2fl/fl-Vav-Cre mice were not fully backcrossed onto the C57BL/6 background, recipient mice and competitor bone marrow cells were from the B6.Cg-Tg(Vav1-Cre)A2Kio/J;B6/126SF1 Mfn2tm3Dcc/Mmucd mixed background mouse strain crossed to B6.SJL-Ptprca Pep3b/BoyJ (CD45.1) to generate a CD45.1+CD45.2+ mixed background mouse. Competitor cells were T-cell depleted using MACS beads. For all competitive transplantation experiments, at least two independent transplants, each with at least 4 recipients per condition of genotype were performed, and result of all recipients pooled for statistical analysis. Power calculation was based on results of the first experiment. In limiting dilution assays, cohorts of recipients received 20 or 50 HSCs together with 2 × 105 competitor cells, allowing calculation of HSC frequency based on the number of non-repopulated mice (<1% donor contribution) using Poisson statistics 15 weeks after reconstitution. For Mfn2 KO single cell transplantation, LT-HSCs were sorted directly into complete media (StemPro34, 100 ng ml−1 SCF, 100 ng ml−1 TPO, 50 ng ml−1 IL-6) and single cells were visually confirmed. Positive single cell wells were combined with 2 × 105 CD45.1 competitor bone marrow cells and transplanted into lethally irradiated CD45.1 recipient mice. Recipients showing ≥ 0.1% CD45.2 donor contribution were considered positive and GM/(B+T) ratios were calculated as previously described for characterizing heterogeneous HSC phenotypes37. In transplantations using WT or Prdm16−/− HSCs (Lin−cKit+Sca1+CD48−Flt3−CD150+) B6.CD45.2 cells were mixed with 2 × 105 freshly isolated B6.CD45.1 bone marrow cells and injected via tail vein into lethally irradiated (two doses of 478 cGy over 3 h using a Rad Source RS-2000 X-ray irradiator (Brentwood, TN)) B6.CD45.1+CD45.2+ F1 hybrid recipients. After 8 to 15 weeks, peripheral blood (PB) and bone marrow were analysed. Lentiviral particles were produced by seeding 293 cells at 7 × 105 per cm2, or PlatE cells (Cell Biolabs, San Diego, CA), in Ultra Culture serum-free media (Lonza, Basel, Switzerland) overnight followed by transfection of each packaging and expression construct (1:1:1) using Trans-It 293 (Mirus, Madison, WI) for 2 h. Media were pooled after 36–48 h, clarified and concentrated by ultracentrifugation (100,000g), resuspended in StemPro-34 media and stored at −80 °C. Virus titre was calculated from transduction of NIH-3T3 fibroblasts serial dilutions of the viral preparation. Sorted LT-HSCs were transduced with ≥ 150 MOI lentivirus particles in the presence of 6 μg ml−1 polybrene (Sigma) and spun at 900g for 20 min at 20 °C. Supernatant was aspirated and replaced with complete media and cultured overnight. Transduction efficiency of cells was confirmed after 24 h. To assess proviral copy number 15 weeks post-transplantation in vivo, splenocytes were harvested and sorted into donor (CD45.2) or competitor (CD45.1) populations and gDNA was isolated as previously described38. Amplification of the proviral WPRE region was achieved using SYBR Green qPCR assay using the primer pair WPREFor: 5′- -3′ and WPRERev: 5′- -3′. Quantification of proviral copies was derived from the linear regression of serial dilutions of viral vector and normalized to input cell number. Sorted or cultured cell populations (2–5 × 103 cells) were lysed in TRIzol LS reagent (Invitrogen, Carlsbad, CA) and RNA was isolated according to manufacturer’s instructions. cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen) and target CT values were determined using inventoried TaqMan probes (Applied Biosystems, Carlsbad, CA, see Supplementary Table 2) spanning exon/exon boundaries and detected using a Viia7 Real Time PCR System (Applied Biosystems). Relative quantification was calculated using the ΔΔC method. To estimate relative copy number of Mfn1 and Mfn2 transcripts (Fig. 4a), copy numbers were derived from the linear regression of serial dilutions of respective cDNA plasmids and normalized to GAPDH-VIC values. To estimate relative copy number of flPrdm16 transcripts (Fig. 4d), a probe was designed to span the SET methyltransferase domain of Prdm16 (exon2/3 junction) and copy number was derived from the linear regression of serial dilutions of respective cDNA plasmids. Another probe (exon 14/15 junction) was used to quantify total Prdm16 copy numbers derived from the linear regression of serial dilutions of respective cDNA plasmids. The values derived from total Prdm16 probe was subtracted from flPrdm16-specific probe to determine sPrdm16 transcript quantity. All values were normalized to relative multiplexed GAPDH-VIC values. Culture of sorted LT-HSCs was carried out using StemPro34 media (Invitrogen) supplemented with 10 mM HEPES and 50 ng ml−1 of recombinant murine SCF, TPO, IL-6 (Peptrotech, Rocky Hill, NJ) and cultured in 5% O at 37 °C. In some experiments, LT-HSCs were cultured in the presence of 500 ng ml−1 VIVIT (Millipore, Billerica, MA) or 30 μM mDivi1 (MolPort, Riga, Latvia). To demonstrate a mitochondrial fusion activity, cell fusion experiments were performed using MEFs as previously described37. Briefly, BacMam baculovirus constructs (Invitrogen) expressing the signalling peptide from cytochrome c fused to either GFP or RFP were transduced separately into MEF cells. Sorted GFP+ and RFP+ MEFs were co-cultured for 24 h and plasma membranes were fused using PEG-1500 (Roche, Basel, Switzerland. Fused cells were cultured in DMEM containing cyclohexamide (Sigma, St. Louis, MO) for 4 h and analysed for colocalization of mitochondrial labels. Early passage Prdm16−/− MEFs were transduced with 10 MOI retrovirus for 72 h and fixed with 4% paraformaldehyde for 10 min. Protein lysates were isolated and chromatin immunoprecipitation was carried out using the ChIP-IT Express Enzyme kit (Active Motif, Carlsbad, CA). Antibodies used for ChIP include anti-Flag and anti-TF2D. Primer probes were designed to span regions of the Mfn2 promoter previously shown to regulate Mfn2 transcriptional activity (see Supplementary Table 3)39. Quantification of precipitated Mfn2 promoter regions were derived from the linear regression of serial dilutions of bone marrow genomic DNA, normalized to input DNA concentration and quantifiable IgG detection was subtracted from sample values. Bone marrow was freshly isolated and lineage depleted with the MACS Lineage Depletion Kit (Miltenyi Biotech, San Diego, CA). Cells were cultured for 30 min in complete medium supplemented with 1 μM Indo-1 prepared as stock supplemented with Pluronic-F127 and incubated at 37 °C for 30 min. Cells were washed and stained for surface markers for 15 min, washed and allowed to rest in for 15 min PBS in PBS with Ca2+. FACS tubes were run at 37 °C in the sample port of the LSRII flow cytometer equipped with a 355 nm excitation laser. Events were collected for 40 s before incubation with 25 μM ATP or 1 μM SDF1 to induce calcium transients. The average ratio, R, of bound/free Indo-1 (405 nm/485 nm emission) before simulation was used to determine baseline values. Identical samples were equilibrated in 10 mM EGTA PBS without Ca2+ to determine R or stimulated with 1 μM ionomycin to determine R . The Indo-1 dissociation constant (K ) was assumed to be 237 nM at 37 °C based on previous studies40. The following equation was then used to relate Indo-1 intensity ratios to [Ca2+] levels; Sorted or cultured haematopoietic populations (2–5 × 103 cells) were collected in complete media and plated on onto MicroWell 96-well glass-bottom plates (Thermo, Waltham, MA) coated with 1ug ml−1 poly-d-lysine. Cells were allowed to adhere for 10 min and fixed with 4% PFA for 15 min. Cells were then permeabilized with 0.1% TritonX-100/PBS for 5 min and blocked with 2% BSA/PBS for 1 h at 4 °C. Cells were incubated with anti-Nfat1 (1:100), anti-Mfn2 (1:200), anti-tubulin (1:200), anti CD150-APC (1:100) or anti-Flag (1:250) (see Supplementary Table 1) overnight, washed and incubated with AlexaFluor secondary antibodies (Invitrogen) for 1 h. Cell nuclei were counterstained with DAPI and mounted with fluorescent mounting media (Vector Labs, Burlingame, CA). Confocal images were acquired with a Zeiss LSM 700 confocal microscope or a Leica DMI 6000B and images were deconvoluted and processed with Leica AF6000 software package. NIH-3T3, WT or Mfn2−/− MEF cells were plated at 2 × 104 cells per cm2 in triplicate overnight and transfected with 500 ng of pGF-Nfat, pGF-CMV or −950/+22 Mfn2-pGL4 reporter construct, 500 ng of cDNA plasmids as indicated and 500 ng of either pSV-βGal or pLVX-IRES-mCherry plasmids with Lipofectamine 3000 according to manufacturer’s instructions for 24 or 48 h. Cells were lysed in reporter lysis buffer (Promega, Madison, WI) and analysed for luciferase activity using BrightGlo luciferase (Promega) and detected on a Synergy H2 plate reader (BioTek, Winooski, VT). To visualize βGal activity, cell lysate was incubated in Buffer Z (1mg ml−1 ONPG, 0.1 M phosphate, pH 7.5, 10 mM KCl, 1 mM βME, 1 mM MgSO ) at 37 °C for 1 h. Absorbance values were measured at 405 nm and used to normalize for transfection efficiency. In WT and Prdm16−/− MEFs, gene reporter luciferase values were normalized to mCherry excitation values. For total cell lysate experiments, MEF cultures were lysed in RIPA buffer, 50 mM Tris pH 7.5, 137 mM NaCl, 0.1% SDS, 0.5% deoxycholate and protease inhibitors (Roche). For subcellular fractionation studies, cells were scraped, washed in PBS. Cell pellets were lysed in 5× packed cell volume (pcv) Buffer A for 10 min on ice and vortexed for 15 s in the presence of 1/10 volume 3% NP-40. Plasma membrane lysis was verified by trypan blue staining. Lysate was spun at 15,000g for 10 min at 4 °C and the cytoplasmic fraction was saved. The remaining nuclear pellet was resuspended in 2.5× pc Buffer C and incubated at 4 °C for 1 h with rotation and spun at 15,000g for 10 min. The nuclear fraction was diluted with 2.5× volume of Nuclear Diluent Buffer and stored at −80 °C. To achieve even fractionation loading, equivalent percentages of nuclear and cytoplasmic fractions were loaded on each gel. All protein samples were denatured in 4× sample buffer at 95 °C and loaded onto 4–12% Bis-Tris SDS–PAGE gradient gels (Invitrogen). Gels were transferred onto 0.22 μm nitrocellulose membrane and stained with Ruby Red (Molecular Probes, Carlsbad, CA) to confirm transfer. Membranes were blocked with 3% non-fat milk or BSA in 0.1%Tween-20/TBS and incubated with anti-Mfn2 (1:200), anti-βGal (1:1,000), anti-Nfat1 (1:250), anti-tubulin (1:1,000), anti-lamin A/C (1:500) and anti-β-actin (1:5,000) overnight (see Supplementary Table 1). Membranes were washed, incubated with HRPO-conjugated secondary antibodies and exposed to X-ray film (Denville) after incubation with Super Signal West Fempto ECL reagent (Pierce). For mitochondrial length measurements, confocal or deconvoluted z-stacks were collected and projected as a z-project in ImageJ (NIH, Bethesda, MD). Individual mitochondria were manually traced, binned into length categories and expressed as percent of cellular mitochondria. The mean ± s.e.m. number of mitochondria falling into each length category collected from ≥ 15 fields (30–50 cells) are expressed. For Nfat nuclear localization quantification, confocal or deconvoluted z-stacks were collected and a 1-μm section in the centre of the cell was projected as a z-project in ImageJ. Nuclear boundaries were constructed using DAPI staining. The ratio of staining within the nuclear boundary to total staining was expressed as percent of Nfat signal. The mean ± s.e.m. for ≥ 10 fields (20–40 cells) are expressed. For immunofluorescence intensity measurements, confocal or deconvoluted z-stacks were collected and projected as a z-project in ImageJ. Thresholds were set based on IgG-stained negative control cells and the integrated density value of each signal per cell was recorded. The mean ± s.e.m. for ≥ 15 fields (30–50 cells) are expressed. For statistical analysis between two groups, the unpaired Student’s t-test was used. When more than two groups were compared, one-way ANOVA was used. Results are expressed as mean ± s.e.m. The Bonferroni and Dunnett multiple comparison tests were used for post-hoc analysis to determine statistical significance between multiple groups. All statistics were calculated using Prism5 (GraphPad, La Jolla, CA) software. Differences among group means were considered significant when the probability value, P, was less than 0.05. Sample size (‘n’) always represents biological replicates. Cochran test was used for exclusion of outliers. No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.


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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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