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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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Human ES cell line H9 (WA-09) and derivatives (SOX10::GFP; SYN::ChR2-EYFP; SYN::EYFP;PHOX2B:GFP;EF1::RFP Ednrb−/−) as well as two independent human iPS cell lines (healthy and familial dysautonomia, Sendai-based, OMSK (Cytotune)) were maintained on mouse embryonic fibroblasts (Global Stem) in knockout serum replacement (KSR; Life Technologies, 10828-028) containing human ES cell medium as described previously7. Cells were subjected to mycoplasma testing at monthly intervals and short tandem repeats (STR) profiled to confirm cell identity at the initiation of the study. Human ES cells were plated on matrigel (BD Biosciences, 354234)-coated dishes (105 cells cm−2) in ES cell medium containing 10 nM FGF2 (R&D Systems, 233-FB-001MG/CF). Differentiation was initiated in KSR medium (knockout DMEM plus 15% KSR (Life Technologies, 10828-028), l-glutamine (Life Technologies, 25030-081), NEAA (Life Technologies, 11140-050)) containing LDN193189 (100 nM, Stemgent) and SB431542 (10 μM, Tocris). The KSR medium was gradually replaced with increasing amounts of N2 medium from day 4 to day 10 as described previously7. For CNC induction, cells were treated with 3 μM CHIR99021 (Tocris Bioscience, 4423) in addition to LDN193189 and SB431542 from day 2 to day 11. ENC differentiation involves additional treatment with retinoic acid (1 μM) from day 6 to day 11. For deriving MNCs, LDN193189 is replaced with BMP4 (10 nM, R&D, 314-BP) and EDN3 (10 nM, American Peptide company, 88-5-10B) from day 6 to day 11 (ref. 3). The differentiated cells are sorted for CD49D at day 11. CNS precursor control cells were generated by treatment with LDN193189 and SB431542 from day 0 to day 11 as previously described7. Throughout the manuscript, day 0 is the day the medium is switched from human ES cell medium to LDN193189 and SB431542 containing medium. Days of differentiation in text and figures refer to the number of days since the pluripotent stage (day 0). For immunofluorescence, the cells were fixed with 4% paraformaldehyde (Affymetrix-USB, 19943) for 20 min, then blocked and permeabilized using 1% bovine serum albumin (BSA) (Thermo Scientific, 23209) and 0.3% Triton X-100 (Sigma, T8787). The cells were then incubated in primary antibody solutions overnight at 4 °C and stained with fluorophore-conjugated secondary antibodies at room temperature for 1 h. The stained cells were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before imaging. For flow cytometry analysis, the cells are dissociated with Accutase (Innovative Cell Technologies, AT104) and fixed and permeabilized using BD Cytofix/Cytoperm (BD Bioscience, 554722) solution, then washed, blocked and permeabilized using BD Perm/Wash buffer (BD Bioscience, 554723) according to manufacturer’s instructions. The cells are then stained with primary (overnight at 4 °C) and secondary (30 min at room temperature) antibodies and analysed using a flow Cytometer (Flowjo software). A list of primary antibodies and working dilutions is provided in Supplementary Table 4. The PHOX2A antibody was provided by J.-F. Brunet (rabbit, 1:800 dilution). Fertilized eggs (from Charles River Farms) were incubated at 37 °C for 50 h before injections. A total of 2 × 105 CD49D-sorted, RFP-labelled NC cells were injected into the intersomitic space of the vagal region of the embryos targeting a region between somite 2 and 6 (HH 14 embryo, 20–25 somite stage). The embryos were collected 36 h later for whole-mount epifluorescence and histological analyses. For RNA sequencing, total RNA was extracted using RNeasy RNA purification kit (Qiagen, 74106). For qRT–PCR assay, total RNA samples were reverse transcribed to cDNA using Superscript II Reverse Transcriptase (Life Technologies, 18064-014). qRT–PCR reactions were set up using QuantiTect SYBR Green PCR mix (Qiagen, 204148). Each data point represents three independent biological replicates. ENC cells from the 11-day induction protocol were aggregated into 3D spheroids (5 million cells per well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF). After 4 days of suspension culture, the spheroids are plated on poly-ornithine/laminin/fibronectin (PO/LM/FN)-coated dishes (prepared as described previously26) in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). The ENC precursors migrate out of the plated spheroids and differentiate into neurons in 1–2 weeks. The cells were fixed for immunostaining or collected for gene expression analysis at days 25, 40 and 60 of differentiation. Mesoderm specification is carried out in STEMPRO-34 (Gibco, 10639-011) medium. The ES cells are subjected to activin A treatment (100 ng ml−1, R&D, 338-AC-010) for 24 h followed by BMP4 treatment (10 ng ml−1, R&D, 314-BP) for 4 days9. The cells are then differentiated into SMC progenitors by treatment with PDGF-BB (5 ng ml−1, Peprotech, 100-14B), TGFb3 (5 ng ml−1, R&D systems, 243-B3-200) and 10% FBS. The SMC progenitors are expandable in DMEM supplemented with 10% FBS. The SMC progenitors were plated on PO/LM/FN-coated culture dishes (prepared as described previously26) 3 days before addition of ENC-derived neurons. The neurons were dissociated (using accutase, Innovative Cell Technologies, AT104) at day 30 of differentiation and plated onto the SMC monolayer cultures. The culture is maintained in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). Functional connectivity was assessed at 8–16 weeks of co-culture. SMC-only and SMC-ENC-derived neuron co-cultures were subjected to acetylcholine chloride (50 μM, Sigma, A6625), carbamoylcholine chloride (10 μM, Sigma,C4382) and KCl (55 mM, Fisher Scientific, BP366–500) treatment, 3 months after initiating the co-culture. Optogenetic stimulations were performed using a 450-nm pigtailed diode pumped solid state laser (OEM Laser, PSU-III LED, OEM Laser Systems, Inc.) achieving an illumination between 2 and 4 mW mm−2. The pulse width was 4 ms and stimulation frequencies ranged from 2 to 10 Hz. For the quantification of movement, images were assembled into a stack using Metamorph software and regions with high contrast were identified (labelled yellow in Supplementary Fig. 5). The movement of five representative high-contrast regions per field was automatically traced (Metamorph software). Data are presented in kinetograms as movement in pixels in x and y direction (distance) with respect to the previous frame. We used the previously described method for generation of tissue-engineered colon11. In brief, the donor colon tissue was collected and digested into organoid units using dispase (Life Technologies, 17105-041) and collagenase type 1 (Worthington, CLS-1). The organoid units were then mixed immediately (without any in vitro culture) with CD49D-purified human ES-cell-derived ENC precursors (day 15 of differentiation) and seeded onto biodegradable polyglycolic acid scaffolds (2-mm sheet thickness, 60 mg cm−3 bulk density; porosity >95%, Concordia Fibres) shaped into 2 mm long tubes with poly-l lactide (PLLA) (Durect Corporation). The seeded scaffolds were then placed onto and wrapped in the greater omentum of the adult (>2 months old) NSG mice. Just before the implantation, these mice were irradiated with 350 cGy. The seeded scaffolds were differentiated into colon-like structures inside the omentum for 4 additional weeks before they were surgically removed for tissue analysis. All mouse procedures were performed following NIH guidelines, and were approved by the local Institutional Animal Care and Use Committee (IACUC), the Institutional Biosafety Committee (IBC) as well as the Embryonic Stem Cell Research Committee (ESCRO). We used 3–6-week-old male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice or 2–3-week-old Ednrbs-l/s-l (SSL/LeJ) mice27 (n = 12, 6 male, 6 female) for these studies. Animal numbers were based on availability of homozygous hosts and on sufficient statistical power to detect large effects between treatment versus control (Ednrbs-l/s-l) as well as for demonstrating robustness of migration behaviour (NSG). Animals were randomly selected for the various treatment models (NSG and Ednrbs-l/s-l) but assuring for equal distribution of male/female ratio in each group (Ednrbs-l/s-l). All in vivo experiments were performed in a blinded manner. Animals were anaesthetized with isoflurane (1%) throughout the procedure, a small abdominal incision was made, abdominal wall musculature lifted and the caecum is exposed and exteriorized. Warm saline is used to keep the caecum moist. Then 20 μl of cell suspension (2–4 million RFP+ CD49D-purified human ES-cell-derived ENC precursors) in 70% Matrigel (BD Biosciences, 354234) in PBS or 20 μl of 70% Matrigel in PBS only (control-grafted animals) were slowly injected into the caecum (targeting the muscle layer) using a 27-gauge needle. Use of 70% matrigel as carrier for cell injection assured that the cells stayed in place after the injection and prevented backflow into the peritoneum. After injection that needle was withdrawn, and a Q-tip was placed over the injection site for 30 s to prevent bleeding. The caecum was returned to the abdominal cavity and the abdominal wall was closed using 4-0 vicryl and a taper needle in an interrupted suture pattern and the skin was closed using sterile wound clips. After wound closure animals were put on paper on top of their bedding and attended until conscious and preferably eating and drinking. The tissue was collected at different time points (ranging from two weeks to four months) after transplantation for histological analysis. Ednrbs-l/s-l mice were immunosuppressed by daily injections of cyclosporine (10 mg kg−1 i.p, Sigma, 30024). The collected colon samples were fixed in 4% paraformaldehyde at 4 °C overnight before imaging. Imaging is performed using Maestro fluorescence imaging system (Cambridge Research and Instrumentation). The tissue samples were incubated in 30% sucrose (Fisher Scientific, BP220-1) solutions at 4 °C for 2 days, and then embedded in OCT (Fisher Scientific, NC9638938) and cryosectioned. The sections were then blocked with 1% BSA (Thermo Scientific, 23209) and permeabilized with 0.3% Triton X-100 (Sigma, T8787). The sections are then stained with primary antibody solution at 4 °C overnight and fluorophore-conjugated secondary antibody solutions at room temperature for 30 min. The stained sectioned were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before they were mounted with Vectashield Mounting Medium (vector, H1200) and imaged using fluorescent (Olympus IX70) or confocal microscopes (Zeiss SP5). Mice are gavaged with 0.3 ml of dye solution containing 6% carmine (Sigma, C1022-5G), 0.5% methylcellulose (Sigma, 274429-5G) and 0.9 NaCl, using a #24 round-tip feeding needle. The needle was held inside the mouse oesophagus for a few seconds after gavage to prevent regurgitation. After 1 h, the stool colour was monitored for gavaged mice every 10 min. For each mouse, total gastrointestinal transit time is between the time of gavage and the time when red stool is observed. The double nickase CRISPR/Cas9 system28 was used to target the EDNRB locus in EF1–RFP H9 human ES cells. Two guide RNAs were designed (using the CRISPR design tool; http://crispr.mit.edu/) to target the coding sequence with PAM targets ~20 base pairs apart (qRNA #1 target specific sequence: 5′-AAGTCTGTGCGGACGCGCCCTGG-3′, RNA #2 target specific sequence: 5′-CCAGATCCGCGACAGGCCGCAGG-3′). The cells were transfected with guide RNA constructs and GFP-fused Cas9-D10A nickase. The GFP-expressing cells were FACS purified 24 h later and plated in low density (150 cells cm−2) on mouse embryonic fibroblasts. The colonies were picked 7 days later and passaged twice before genomic DNA isolation and screening. The targeted region of EDNRB gene was PCR amplified (forward primer: 5′-ACGCCTTCTGGAGCAGGTAG-3′, reverse primer: 5′-GTCAGGCGGGAAGCCTCTCT-3′) and cloned into Zero Blunt TOPO vector (Invitrogen, 450245). To ensure that both alleles (from each ES cell colony) are represented and sequenced, we picked 10 bacterial clones (for each ES cell clone) for plasmid purification and subsequent sequencing. The clones with bi-allelic nonsense mutations were expanded and differentiated for follow-up assays. The ENC cells are plated on PO/LM/FN coated (prepared as described previously26) 96-well or 48-well culture plates (30,000 cm−2). After 24 h, the culture lawn is scratched manually using a pipette tip. The cells are given an additional 24–48 h to migrate into the scratch area and fixed for imaging and quantification. The quantification is based on the percentage of the nuclei that are located in the scratch area after the migration period. The scratch area is defined using a reference well that was fixed immediately after scratching. Migration of cells was quantified using the open source data analysis software KNIME29 (http://knime.org) with the ‘quantification in ROI’ plug-in as described in detail elsewhere30. To quantify proliferation, FACS-purified ENC cells were assayed using CyQUANT NF cell proliferation Assay Kit (Life Technologies, C35006) according to manufacturer’s instructions. In brief, to generate a standard, cells were plated at various densities and stained using the fluorescent DNA binding dye reagent. Total fluorescence intensity was then measured using a plate reader (excitation at 485 nm and emission detection at 530 nm). After determining the linear range, the CD49D+ wild-type and Ednrb−/− ENC precursors were plated (6,000 cell cm−2) and assayed at 0, 24, 48 and 72 h. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To monitor the viability of wild-type and Ednrb−/− ENC precursors, cells were assayed for lactate dehydrogenase (LDH) activity using CytoTox 96 cytotoxicity assay kit (Promega, G1780). In brief, the cells are plated in 96-well plates at 30,000 cm−2. The supernatant and the cell lysate is collected 24 h later and assayed for LDH activity using a plate reader (490 nm absorbance). Viability is calculated by dividing the LDH signal of the lysate by total LDH signal (from lysate plus supernatant). The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. The chemical compound screening was performed using the Prestwick Chemical Library. The ENC cells were plated in 96-well plates (30,000 cm−2) and scratched manually 24 h before addition of the compounds. The cells were treated with two concentrations of the compounds (10 μM and 1 μM). The plates were fixed 24 h later for total plate imaging. The compounds were scored based on their ability to promote filling of the scratch in 24 h. The compounds that showed toxic effects (based on marked reduction in cell numbers assessed by DAPI staining) were scored 0, compounds with no effects were scored 1, compounds with moderate effects were scored 2, and compounds with strong effects (that resulted in complete filling of the scratch area) were scored 3 and identified as hit compounds. The hits were further validated to ensure reproducibility. The cells were treated with various concentrations of the selected hit compound (pepstatin A) for dose response analysis. The optimal dose (10 μM based on optimal response and viability) was used for follow-up experiments. For the pre-treatment experiments, cells were CD49D purified at day 11 and treated with pepstatin A from day 12 to day 15 followed by transplantation into the colon wall of NSG mice. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To inhibit BACE2, the ENC precursors were treated with 1 μM β-secretase inhibitor IV (CAS 797035-11-1; Calbiochem). To knockdown BACE2, cells were dissociated using accutase (Innovative Cell Technologies, AT104) and reverse-transfected (using Lipofectamine RNAiMAX-Life Technologies, 13778-150) with an siRNA pool (SMARTpool: ON-TARGETplus BACE2 siRNA, Dharmacon, L-003802-00-0005) or four different individual siRNAs (Dharmacon, LQ-003802-00-0002, 2 nmol). The knockdown was confirmed by qRT–PCR measurement of BACE2 mRNA levels in cells transfected with the BACE2 siRNAs versus the control siRNA pool (ON-TARGETplus Non-targeting Pool, Dharmacon, D-001810-10-05). The transfected cells were scratched 24 h after plating and fixed 48 h later for migration quantification. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. Data are presented as mean ± s.e.m. and were derived from at least three independent experiments. Data on replicates (n) is given in figure legends. Statistical analysis was performed using the Student’s t-test (comparing two groups) or ANOVA with Dunnett test (comparing multiple groups against control). Distribution of the raw data approximated normal distribution (Kolmogorov–Smirnov normality test) for data with sufficient number of replicates to test for normality. Survival analysis was performed using a log-rank (Mantel–Cox) test. Z-scores for primary hits were calculated as Z = (x − μ)/σ, in which x is the migration score value and is 3 for all hit compounds; μ is the mean migration score value, and σ is the standard deviation for all compounds and DMSO controls (n = 224).


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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.


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HBx fused at its carboxy (C) terminus via a 10-amino-acid linker (HMRSRSGLET) either to wild-type DDB1 or to the CUL4-binding-defective DDB1 mutant has been described8, 28. The SV5-V–DDB1 fusions were generated by replacing the sequence encoding HBx within the HBx–DDB1 fusions by the full-length SV5-V coding region13. Unless fused to DDB1, all viral proteins contain an amino (N)-terminal GFP tag and have been described6. For TAP, the proteins carried an N-terminal Flag tag followed by two Strep-tag II sequences (FS-tag)29. For ChIP analysis, the full-length NSE4a coding region was amplified by PCR from a Namalwa B cell complementary DNA (cDNA) library and fused N-terminal to three tandem copies of the HA epitope. Proteins were expressed from a stably integrated tetracycline-inducible pTRE2hyg plasmid vector (Clontech) in Fig. 1b and Extended Data Fig. 1a, b, from the episomal Epstein–Barr virus-based expression vector EBS-PL30 in Fig. 1c and Extended Data Fig. 1d, from the tetracycline-inducible lentiviral vector pINDUCER20 (ref. 31) (a gift from S. Elledge) in Fig. 2c and Extended Data Fig. 2e, and from the lentivirus vector pWPT6, 7 in all the other figures. The luciferase reporter genes were constructed into pcDNA3 (pGL3 in the case of HBV Enhancer I6) for transient transfection and into the self-inactivating lentiviral vector pWPT (pWPXL in the case of HBV Enhancer I6) for chromosomal integration. In Extended Data Fig. 3c, the EF1α episomal reporter was delivered using an integrase-defective (D116A) lentiviral vector32. The short hairpin RNAs (shRNAs) used for Smc5 and Smc6 knockdown were cloned into the miR30 context as 116-nucleotide XhoI–EcoRI fragments into pINDUCER10 (ref. 31) and/or pGIPZ (Open Biosystems) lentiviral vectors. The sequences were as follows, with the hairpin sequence in capital letters and flanking miR30 sequences shaded in bold type: shSmc5(1), gagcgGTGAGGTGAAAGAAGTGTTTCTtagtgaagccacagatgtaAGAAACACTTCTTTCACCTCATtgcct; shSmc5(2), gagcgGTGCGAAACTTGTTACCGAATTtagtgaagccacagatgtaAATTCGGTAACAAGTTTCGCATtgcct; shSmc6(1), gagcgGTGAGCAGCTTTGTAAACGAATtagtgaagccacagatgtaATTCGTTTACAAAGCTGCTCATtgcct; shSmc6(2) (from Thermo Scientific (V3LHS_325916), gagcgCAGAGACAGTGCTACTAATCAAtagtgaagccacagatgtaTTGATTAGTAGCACTGTCTCTAtgcct. The constructs used were shSmc5(2) and shSmc6(1) in Fig. 2d, shSmc5(1) and shSmc6(2) in Extended Data Fig. 2f, and shSmc6(2) in Extended Data Fig. 2g. The Cardif shRNA construct in pINDUCER10 was a gift from D. Garcin. The GAPDH shRNA construct in pGIPZ was purchased from Thermo Scientific (RHS4371). Expression was from pINDUCER10 in Fig. 2d and Extended Data Fig. 2f, g and from pGIPZ in Fig. 3c, d. Details of plasmid constructions are available upon request. The sequences of the siRNAs used in the other figures can be found in Supplementary Table 2. The human hepatoma cell lines HepG2 (ATCC HB-8065), HepG2tet-on (ref. 33), and derivatives were grown at 37 °C under 5% CO in modified Eagle’s medium (MEM; Life Technologies or Sigma-Aldrich) supplemented with 10% (vol/vol) fetal calf serum (Gibco), 1% (v/v) penicillin/streptomycin solution, 2 mM l-glutamine, 1 mM sodium pyruvate, and 0.1 mM MEM non-essential amino acids solution (all from Life Technologies). In Extended Data Fig. 6d, HepaRG cells were cultured, induced to differentiate, and infected with HBV as described34. The cell lines were not authenticated or tested for Mycoplasma contamination because of their direct purchase from ATCC or low passage number. The stable HepG2 cell lines used in Fig. 1b and Extended Data Fig. 1a, b that expressed the tetracycline-inducible transactivator rtTA and various FS-tagged proteins from a tetracycline-responsive promoter were established as follows. The HepG2tet-on parental cell line33 was plated at a density of 1 × 105 cells per 6-cm dish and transfected using FuGENE HD (Roche) with the relevant constructs cloned into pTRE2hyg (Clontech), which carries a hygromycin resistance marker. At day 3 after transfection, cells were selected in medium containing 250 μg ml−1 hygromycin B (PAA Laboratories) and 100 μg ml−1 G418 (Geneticin, Gibco)33. Resistant colonies were picked and expanded in 48-well plates under the same selection conditions. Individual clones were assayed by reverse transcription (RT)-PCR and western blot analyses for transgene expression in the presence or absence of 2 μg ml−1 doxycycline (Sigma Aldrich). The HepG2 cells in Fig. 2c expressing the doxycycline-inducible HBx–DDB1 fusions from pINDUCER20 lentiviral vector carrying a neomycin resistance marker were obtained by selection for 2 days with 1 mg ml−1 G418 (Promega) starting at day 3 after transduction. The HepG2 cells in Fig. 2d and Extended Data Fig. 2f, g expressing doxycycline-inducible shRNAs against Smc5, Smc6, or Cardif from pINDUCER10 lentiviral vector were obtained by selection with 10 μg ml−1 puromycin (Calbiochem) for 4 days starting at day 3 after transduction. The resulting puromycin-resistant cells were then cultured for 2 weeks in the absence or presence of 2.5 μg ml−1 doxycycline before transduction of GFP or GFP–HBx to maximally induce shRNA expression. Fluorescence microscopy indicated that >90% of the cells were expressing turboRFP (tRFP) from the tRFP-shRNA cassette31. The HepG2.2.15 (ref. 4), HepAD38 (ref. 35), and HepG2-H1.3x- (ref. 4) stable cell lines used to produce HBV virions carrying either a wild-type genome or a genome bearing a defective HBx gene have been described. They were maintained in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12) complemented with 10% fetal calf serum, 1% non-essential amino acids, 50 μg ml−1 kanamycin, and 200 μg ml−1 geneticin. Transfection of plasmid DNA in HepG2 cells was performed using X-tremeGENE HP DNA Transfection Reagent (Roche) following the manufacturer’s instructions. Transfection of siRNA duplexes in HepaRG cells was performed using DharmaFECT-1 (Thermo Scientific) transfection reagent and in PHHs using Lipofectamine RNAiMax (Life Technologies). Recombinant lentivirus production and transduction of HepG2 cells were performed as described previously7, 36. For PHH transduction, lentivirus supernatants were concentrated by ultracentrifugation at 130,000 g for 1 h in a SW32 Beckman rotor at 4 °C and transduction was performed overnight in the presence of 10 ng ml−1 of EGF37. For luciferase reporter gene assay and western blot analysis, cells were typically seeded at a density of about 6 × 105 cells per 30 mm diameter well (1 × 105 cells per square centimetre) and transfected the next day with 30 ng of reporter plasmid DNA and 2 μg of empty EBS-PL vector. For the ChIP experiment in Fig. 2e, 3 or 30 ng of reporter plasmid were used with similar results. In Fig. 2d and Extended Data Fig. 2f, g, cells that had been cultured in the absence or presence of doxycyclin for 2 weeks to induce shRNA expression from pINDUCER10 were first transduced and the next day transfected with, respectively, lentiviral and plasmid constructs carrying the Renilla and firefly luciferase reporter genes as indicated in the figures. Cells were trypsinized the following day, washed with phosphate-buffered saline (PBS), replated in 6- or 24-well dishes at a density of about 6 × 104 cells per square centimetre and transduced 6 h after replating with lentiviral vectors encoding GFP or the GFP-tagged viral proteins. In Fig. 2b, dimethylsulphoxide (DMSO) or 5 μM MLN4924 (Active Biochem) was added 6 h before transduction and culture medium was replaced daily with fresh medium containing (or not) MLN4924 for 3 days. In Fig. 2c, d and Extended Data Fig. 2f, g, cells were constantly grown in the absence or presence of 2 μg ml−1 doxycycline. Cell lysates were prepared 6 days later (3 days in Fig. 2b) for luciferase assay and western blot analysis. Luciferase activities were measured using the Luciferase Assay System (Promega) or Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The activities were normalized to protein concentrations as measured by the Bradford assay (Bio-Rad). The two-step purification scheme was adapted from ref. 29. The HepG2tet-on cell lines conditionally expressing FS-tagged proteins from a doxycycline-inducible promoter were seeded on thirty 15-cm dishes and allowed to reach 90% confluence. Expression of the FS-tagged proteins was then induced by addition of doxycycline (2 μg ml−1) for 48 h. Cells were rinsed once with 20 ml PBS (per plate) and scraped off after incubation on ice for 20 min in 1.5 ml lysis buffer (50 mM HEPES/KOH, pH 7.5, 150 mM NaCl, 5 mM KCl, 5 mM MgCl , 50 μM ZnCl , 0.1% IGEPAL and protease inhibitor cocktail from Sigma). The lysates were pooled, supplemented with glycerol (10% final), and clarified by centrifugation at 20,000g for 20 min at 4 °C in a Sorvall HB-6 rotor. Supernatants were collected, passed through a 0.45 μm Millipore PVDF filter, and mixed in a 50 ml Falcon tube with a 100 μl packed bead volume of Strep-Tactin Sepharose (IBA) washed with lysis buffer. After incubation for 2 h at 4 °C under constant rotation, the suspension was poured into a 10 ml disposable column (Bio-Rad). After sedimentation, the resin was washed twice with 10 ml lysis buffer containing 10% glycerol. The bound proteins were released by incubating the resin with 10 ml of lysis buffer supplemented with 10% glycerol and 2.5 mM desthiobiotin (IBA 2× Tactin Elution Buffer) for 10 min on ice. The eluted material was directly mixed with a 100 μl packed volume of washed anti-Flag Sepharose beads (Sigma) and incubated with rotation for 1 h at 4 °C in a sealed 10 ml disposable column (Bio-Rad). Subsequently, the column was drained by gravity and the resin washed five times with 1 ml lysis buffer adjusted to 10% glycerol. Bound proteins were recovered by adding five times sequentially 200 μl lysis buffer supplemented with 200 μg ml−1 Flag peptide (Sigma) and gently mixing for 5 min on ice. The eluted material from three such purifications was pooled and precipitated with acetone (80% final concentration). After centrifugation at 4 °C in an Eppendorf microcentrifuge, the protein pellet was air-dried, resuspended in 1× Laemmli sample buffer, and run into a 4% SDS–PAGE stacking gel. The protein band was excised and processed for mass spectrometry analysis using an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Listed in Fig. 1b are proteins identified with 99% certainty and represented by at least two peptides ascertained at a 95% confidence level. In Fig. 1c, extracts from 6 × 106 HepG2 cells seeded on a 10-cm culture dish were prepared 2 days after transfection with 2 μg of EBS-PL expression plasmids. The experiment in Extended Data Fig. 1d was performed in the same way except that the transfected cells were selected in medium containing 200 μg ml−1 hygromycin B (PAA Laboratories) and extracts were prepared from a 15-cm dish of confluent cells. Proteins were purified by incubation with 25 μl packed bead volume of Strep-Tactin Sepharose (IBA). The cell cycle analysis in Extended Data Fig. 3a was performed starting with 1.5 × 106 HepG2 cells transduced with GFP or GFP–HBx and cultured as above. Cells were harvested, washed once with PBS, and recovered by centrifugation at 500g for 5 min. Cells were fixed in 1 ml ice-cold 70% ethanol added dropwise while vortexing and incubated for 30 min on ice. The fixed cells were pelleted by centrifugation at 800g for 15 min, washed twice with PBS and directly resuspended in 50 μl PBS supplemented with 100 μg ml−1 RNase A (Roche). Cellular DNA was stained by addition of 600 μl of propidium iodide (Sigma Aldrich; 50 μg ml−1 in PBS) and incubation for 10 min at room temperature (20–25 °C) and overnight at 4 °C. Samples were analysed using a BD Accuri C6 flow cytometer. With the exception of Fig. 4b and Extended Data Fig. 6g (see below), western blot analysis was performed as described38 except that cells were disrupted in 2% SDS in water and protein concentration was estimated using the BCA Protein Assay (Novagen). The membranes were probed with 1:5,000 mouse anti-Flag antibodies (Sigma A2220) to detect the FS-tagged HBx–DDB1 and SV5-V–DDB1 fusion proteins, 1:5,000 mouse monoclonal anti-GFP (Roche 11814460001) to detect GFP–HBx in Extended Data Fig. 2a, b, 1:5,000 mouse monoclonal anti-HA (clone 16B12, Covance) to detect HA-Nse4 in Fig. 2e, 1:2,000 rabbit polyclonal antibodies against Smc5 or Smc6 (ref. 39) (a gift from A. R. Lehmann), 1:1,000 rabbit polyclonal anti-Nse4 (Abgent AP9909A), 1:2,000 rabbit polyclonal anti-Cardif/MAVS (Enzo Life Sciences ALX-210-929), 1:500 mouse monoclonal anti-STAT1 (BD Transduction Laboratories G16920), 1:500 goat polyclonal anti-DDB1 (Everest Biotech), 1:500 rabbit polyclonal anti-HBc (a gift from M. A. Petit), 1:500 mouse monoclonal anti-ubiquitin (Santa Cruz SC-8017), 1:10,000 mouse monoclonal anti-α-tubulin (Sigma-Aldrich T5168), or 1:10,000 mouse monoclonal anti-GAPDH (Sigma-Aldrich G8795) antibodies. Horseradish-peroxidase-conjugated sheep anti-rabbit or anti-mouse, or bovine anti-goat IgG (Amersham Biosciences, 1:5,000), were used as secondary antibodies, and detection was performed with ECL (Pierce). In Fig. 4b, flash-frozen liver tissue from uPA-SCID mice re-populated with human hepatocytes was homogenized in RIPA buffer containing a broad spectrum protease inhibitor cocktail (Fisher Scientific PI-78430) using a Qiagen Tissue Lyser for mechanical tissue disruption. The resulting homogenates were clarified by centrifugation for 10 min at 4 °C in an Eppendorf 5415D microcentrifuge equipped with a fixed-angle rotor (F45-24-11) at 15,996g. Protein concentration was determined using the Bradford Protein assay (Bio-Rad). The membranes were probed with 1:1,000 mouse monoclonal anti-human Smc6 (Abgent AT3956A), 1:1,000 mouse anti-HBsAg (International ImmunoDiagnostics 1113), and 1:1,000 monoclonal mouse anti-human CK-18 (Dako M701029-2). IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse IgG (Licor, 1:5,000) were used as secondary antibodies. Blots were visualized using an Odyssey Infrared Imaging System (Licor). The species specificity of the anti-human Smc6 and CK-18 antibodies was confirmed by western blot detection of Smc6 and CK-18 in HepG2 cells and PHHs but not in murine hepatocyte AML12 cells (ATCC CRL-2254, data not shown). In Extended Data Fig. 6g, PHHs were lysed in RIPA buffer supplemented with 1× protease inhibitor cocktail (ThermoScientific), scraped from the plate, and sonicated for 10 s to break up cellular debris. Protein concentration was estimated using a BCA Protein Assay (Novagen). The membranes were probed with 1:1,000 mouse monoclonal anti-Smc5 (Bethyl Laboratories A300-236A), 1:1,000 mouse monoclonal anti-human Smc6 (Abgent AT3956A), 1:1,000 rabbit polyclonal anti-DDB1 (Cell Signaling 5428), and 1:1,000 mouse monoclonal anti-GAPDH (Novus Biologicals G8795). Secondary antibodies and detection were as in Fig. 4b. In Extended Data Fig. 2c, total cellular RNA was extracted from HepG2 cells using TRIzol reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. RNA was treated with RNase-free DNase (Promega) in the presence of 1 U μl−1 RNAsin (Promega). After phenol/chloroform extraction and ethanol precipitation, 1 μg of RNA was reverse transcribed using MLV reverse transcriptase (Promega) and an oligo(dT)15 primer. Quantification by real-time PCR was performed as described6. The PCR values were normalized against those obtained for the TBP gene to correct for variation between samples. In Extended Data Fig. 6e, f, i, total cellular RNA was isolated from PHHs cultured in 96-well plates using an RNeasy 96 Kit (Qiagen) following the manufacturer’s instructions. Real-time RT–PCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Invitrogen Life Technologies) following the manufacturer’s instructions. The PCR values were normalized against those obtained for the β-actin gene to correct for variation between samples. All oligonucleotide primer sets were manufactured by Life Technologies. Northern blot analysis in Fig. 3c and Extended Data Fig. 6c was performed on total RNA isolated using TRIzol Reagent (Gibco-Invitrogen) and treated with RNase-Free DNase I (Ambion, Life Technologies). The RNA (10 μg per sample) was denatured by glyoxal treatment and separated on a 1% agarose gel using a NorthernMax-Gly kit (Ambion, Life Technologies). After capillary transfer to Hybond N+ membranes (Amersham), the RNA was fixed on the membrane by ultraviolet cross-linking and hybridized with a 32P-labelled full-length HBV genomic DNA or 28S ribosomal RNA oligonuclotide probes. The HBV producing HepG2 cell lines4 were grown to confluence in DMEM/F12 as described above. When confluence was reached, cells were maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Williams’ E medium (Invitrogen Life Technologies) supplemented with 5% fetal calf serum, 7 × 10−5 M hydrocortisone hemisuccinate, 5 μg ml−1 insulin, and 1% DMSO. HBV-containing supernatants were collected every 2 days for 10 days. The supernatants were pooled, filtered through a 0.22 μm filtration unit (Merck Millipore), and concentrated by overnight precipitation with PEG 8000 (5% final) and centrifugation at 4 °C for 1 h at 6,000g. The viral pellet was resuspended in 1/50 of the original culture volume in PBS and sedimented at 4 °C through a 20 ml 10–20% sucrose gradient in PBS at 130,000g for 16 h in a SW32 Beckman rotor. The final pellet was resuspended in 1/100 of the original volume in William’s E medium supplemented with 2% DMSO and 0.1% SVF and stored in aliquots at −80 °C. Infectious virus titre was estimated by real-time PCR quantification of the viral DNA recovered by immunoprecipitation with an excess of mouse monoclonal antibodies against the large envelope protein (preS1)40. Real-time PCR was performed using the SYBR Green PCR Master Mix (Roche Applied Science) and a LightCycler 480 system (Roche Applied Science) as described34. HBV virion production from HepAD38 cells was as follows. Cells were grown to confluence in DMEM/F12 supplemented with 10% fetal bovine serum (HyClone, Thermo Scientific), 1% non-essential amino acids, 50 μg ml−1 kanamycin, 200 μg ml−1 geneticin (all from Gibco Life Technologies), and 0.3 μg ml−1 tetracycline (Sigma-Aldrich). Once confluence was reached, the medium was exchanged for identical medium as above, but lacking tetracycline. After 10 days, the virus-containing medium was collected every 3–4 days for 21 days and stored at −80 °C. Subsequently, the aliquots were thawed, precipitated overnight at 4 °C using 6% PEG-8000 (Promega), and centrifuged at 4 °C for 15 min at 1,500g. Viral DNA was isolated using a DNeasy Blood & Tissue Kit (Qiagen). Viral titre was determined by real-time PCR. PHHs were isolated from resected normal human liver tissue using a two-step perfusion method and cultured as described4, 41. PHHs were infected with PCR-normalized HBV virus stocks at a multiplicity of infection of 200–400 viral genome equivalents per cell34. The experiments in Fig. 3b, e and Extended Data Fig. 6e–i were performed using PHHs purchased from Life Technologies and maintained in William’s E medium with added supplements as specified by the vendor. Cells were infected 24 h after plating on collagen-coated 96-well plates (BD Biosciences) with HepAD38-derived HBV virions at 500 viral genome equivalents per cell. In Extended Data Fig. 6g, cells were cultured on collagen-coated six-well plates (BD Biosciences). All animal work was performed by Phoenix Bio, in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Phoenix Bio. Nine male uPA-SCID mice were transplanted at 16–23 days old with 1 × 106 PHHs from a single healthy donor as described42. After 10–13 weeks, six of these mice were infected by intraperitoneal injection with 5 × 105 genome equivalents of cell-culture-derived HBV genotype C. These animals were killed and serum and liver specimens were collected for measurement of HBV infection 14 weeks later. Serum HBV DNA reached a titre of >1.5 × 107 copies per millilitre and serum HBsAg levels were ≥3.2 × 102 IU ml−1 in all infected animals. As a control, three of the nine mice were left uninfected. These animals were killed at 15 weeks after transplantation and then processed identically to the HBV-infected animals. The amounts of HBe and HBs antigens present in control and HBV-infected PHH culture media were determined at the indicated times by ELISA using Monolisa HBe Ag-Ab PLUS (Bio-Rad) and Monolisa anti-HBs PLUS (Bio-Rad) kits and a Chemiluminescence Immunoassay (Autobio Diagnostics) kit. In Fig. 3e and Extended Data Fig. 6h, ELISA was performed using an HBeAg EIA (International Immunodiagnostics) kit and a SpectraMax M5 reader (Molecular Devices). Purified HBeAg was used as standard. The proportion of PHHs infected with HBV in Fig. 3d and Extended Data Fig. 5 was estimated by confocal immunofluorescence microscopy for HBsAg expression34. PHHs were cultured and infected on collagen-I-coated glass slides. At 9–12 days after infection, cells were fixed with 3% paraformaldehyde in PBS and permeabilized with 0.1% saponin in PBS for 30 min at room temperature. Cells were stained with mouse monoclonal anti-HBs antibodies (a gift from M. A. Petit) diluted 1:200 in PBS containing 0.1% saponin and 3% bovine serum albumin (BSA). After washing in the same buffer, bound antibodies were detected using Alexa-Fluor-647-conjugated goat anti-mouse secondary antibodies (Invitrogen). After additional washes, the slides were mounted with fluorescence mounting medium (ProLong Gold, Life Technologies) and observed under a Leica SP5 X confocal microscope. Image analysis used ImageJ software. Confocal analysis of Smc6 expression in HBV-infected and uninfected PHHs in Fig. 3b and Extended Data Fig. 7 was performed as follows. PHHs were seeded onto rat tail collagen-I-coated glass coverslips (Corning BioCoat 354089) at a density of 1.2 × 106 cells per well of a 6-well dish and allowed to adhere overnight. Cells were then mock-infected or infected at 500 viral genome equivalents per cell with wild-type HBV derived from HepAD38 cells. On day 13 after infection, the cells were washed twice with PBS and fixed in freshly prepared 4% paraformaldehyde (Electron Microscopy Services RT-15710) in PBS for 10 min at room temperature. After three washes in PBS, the cells were permeabilized in 1% TX-100 in PBS for 10 min at room temperature. Coverslips were incubated overnight at 4 °C in 3% normal goat serum (Jackson ImmunoResearch Laboratories 005-000-121) diluted in PBS to quench non-specific background. Coverslips were then inverted onto 100 μl droplets containing mouse anti-human Smc6 (Abgent AT3956a, 1:500) and polyclonal rabbit anti-HBcAg (Dako B0586, 1:1,600) diluted in 3% normal goat serum, and incubated at room temperature for 1 h. As a control, cells were reacted in parallel with purified mouse IgG2a isotype-matched control (Life Technologies MG2a00). After eight washes in PBS, coverslips were inverted onto 100 μl droplets of highly cross-adsorbed Alexa Fluor 488 goat-anti-mouse (Molecular Probes A11029) or Alexa Fluor 594 goat-anti-rabbit (Molecular Probes A11037) secondary antibodies diluted 1:200 in 3% normal goat serum, and incubated for 1 h at room temperature. Coverslips were washed eight times in PBS and rinsed in double-distilled water before mounting onto glass slides using Vectashield DAPI-containing hardening mounting medium (Vector Laboratories H-1500). Imaging was performed using an upright Zeiss LSM780 confocal system equipped with a ×63 objective lens (NA 1.4) and Zen software. All images within each sample set were captured using identical confocal settings. Images were adjusted for brightness and contrast using Adobe Photoshop CS6. For immunofluorescence analysis of humanized mouse liver tissues, flash-frozen liver tissues was cryosectioned at a thickness of 5 μm onto microscope slides, fixed for 10 min in ice-cold 4% paraformaldehyde in PBS, and then washed three times in PBS. After one wash in Tween 20-containing AutoWash (BioCare Medical TWB945M), tissue sections were incubated with Rodent Block M solution (BioCare Medical RBM961L) for 30 min at room temperature, then stained overnight at 4 °C in a humidified chamber with mouse monoclonal anti-human Smc6 (Abgent AT3956a, 1:200) and rabbit polyclonal anti-HBsAg (Virostat 1811, 1:500) antibodies diluted in a 1:1 solution of Rodent Block M (BioCare Medical RBM961) and Renoir Red diluent (BioCare Medical PD904M). In parallel, re-population of liver tissue with human hepatocytes was evaluated by staining representative sections from each animal with goat anti-human albumin polyclonal antibodies (Bethyl A80-129A, 1:200), whose species specificity has been demonstrated43, and for selected animals by co-staining with mouse monoclonal anti-human cytokeratin-18 antibody (Dako M7010, 1:25). Mouse IgG1 (Dako X0931) diluted in Dako Antibody Diluent S0809 served as a negative control (see Extended Data Fig. 9). After three 5 min washes in AutoWash buffer, tissue sections were reacted for 1 h at room temperature with Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 594 donkey anti-rabbit secondary antibodies diluted 1:1,000 in PBS. Coverslips were applied using Vectashield DAPI-containing embedding medium (Vector Laboratories H-1500). Images were acquired using a ×40 objective lens (Fig. 4a) or a ×20 objective lens (Extended Data Fig. 8) and an inverted epifluorescence microscope (Leica DM LB) and arranged using Adobe Photoshop CS6. Note that imaging in Fig. 4a and Extended Data Fig. 8a was limited to areas completely re-populated with human hepatocytes, which were readily distinguished from poorly engrafted areas of mouse liver tissue on the basis of cellular morphology Extended Data Fig. 8b. The ChIP analysis in Fig. 2e was performed using chromatin extracted from about 7 × 106 HepG2 cells cultured in 100 mm diameter wells as above and expressing HA-Nse4 from a lentiviral vector. In Fig. 3f, freshly prepared PHHs were seeded into 75 cm2 collagen-coated tissue culture flasks (107 cells per flask). Two days later, cells were infected with wild-type or HBx-mutant HBV particles. After culture for the indicated periods, cells were fixed with 1% formaldehyde (Sigma Aldrich 47608) for 10 min at 37 °C before quenching with 330 mM glycine. Cells were rinsed twice with ice cold PBS containing EDTA-free protease inhibitors (Roche) and 5 mM Na-butyrate, scraped off in 1.5 ml of the same buffer and collected by centrifugation. Cells were resuspended and lysed for 10 min at 4 °C in 1 ml Nuclear Extraction buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1% NP-40) supplemented with protease inhibitor cocktail (Roche). The nuclei were recovered by centrifugation at 500g for 5 min at 4 °C in an Eppendorf fixed-angle rotor (FA-45-18-11) and washed once in the same buffer. Nuclei were resuspended in 100 μl FA-lysis buffer (50 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 1% SDS and incubated for 10 min at room temperature. After addition of another 100 μl FA-lysis buffer, the mixture was transferred to 1.5 ml bioruptor microtubes (Diagenode), and sonicated using a Diagenode Bioruptor Pico water bath sonicator (ten cycles of 15 s on and 30 s off at high setting). The sonicated lysates were clarified by centrifugation at 16,000g for 10 min and mixed in 800 μl FA-lysis buffer supplemented with protease inhibitors with 40 μl packed bead volume of protein A-Sepharose CL-4B (GE Healthcare) coupled to 4 μl anti-HA antibodies (clone 16B12, Covance) or 6 μl anti-Nse4 antibodies (Abgent AP9909A) and pre-incubated in FA-lysis buffer with sonicated salmon sperm DNA and BSA. After an overnight incubation at 4 °C under constant rotation, the beads were sedimented by brief centrifugation and the supernatant either discarded or in Extended Data Fig. 3c directly mixed and incubated overnight at 4 °C with anti-Nse4 affinity beads as above. The beads were washed twice with 1 ml FA-lysis buffer, twice with FA-lysis buffer adjusted to 500 mM NaCl and 0.5% sodium deoxycholate, and twice with 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, 250 mM LiCl, 1% NP-40, and 1% sodium deoxycholate. Bound protein–DNA complexes were released from the HA- and Nse4-affinity resins by incubation for 10 min at 65 °C in 200 μl buffer containing 100 mM Tris-HCl (pH 8.0), 1% SDS, 10 mM EDTA, and 10 mM EGTA. After addition of 200 μl Tris-EDTA (pH 8.0) and 60 μg proteinase K (Bioworld Technology), DNA crosslinks were reversed by overnight incubation at 65 °C. Samples were extracted twice with phenol–chloroform, once with chloroform, and then ethanol precipitated and resuspended in Tris-EDTA buffer. The input DNA treated identically and the recovered DNA were quantified in two separate real-time PCR runs using the KAPA SYBR FAST qPCR Kit Master Mix (2X) Universal (Kapa Biosystems) and the Bio-Rad CFX96 Real-time PCR System. The values were calculated as the ratios between the ChIP signals and the respective input DNA signals. In Fig. 3e, primers specific for the covalently closed circular HBV DNA (cccDNA)14 were used. Sequences of the oligonucleotide primers are given in Supplementary Table 2. No statistical methods were used to predetermine sample size. Statistical significance was tested using a one-tailed, paired t-test (for two sample comparisons) or one-way ANOVA with Dunnett’s multiple comparison correction of log-transfomed data (for multiple comparisons). A value of P < 0.05 was considered significant. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. PHHs were isolated from liver specimens resected from patients undergoing partial hepatectomy (provided by M. Rivoire). Approval from the local and national ethics committees (French Ministry of Research and Education numbers AC-2013-1871 and DC-2013-1870) and informed consent from patients were obtained.


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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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