News Article | October 28, 2015
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. Complementary DNA (cDNA) for human GSDMD was amplified from reverse-transcribed cDNA of HT-29 cells; cDNAs for human GSDMB, human GSDMC and mouse Gsdma3 were synthesized by our in-house gene synthesis facility; cDNAs for human GSDMA and mouse Gsdmd were obtained from Vigene Biosciences (CH892815) and OriGene (MC202215), respectively. The gasdermin cDNAs were inserted into a modified pCS2-3×Flag vector for transient expression in 293T cells and the pWPI lentiviral vector with an N-terminal 2×Flag–HA tag or the FUIGW vector with an N-terminal Flag tag for stable expression in HeLa and iBMDM cells. For recombinant expression in E. coli, the cDNAs were cloned into a modified pET vector with an N-terminal SUMO tag. Truncation mutants of the gasdermins were constructed by the standard PCR cloning strategy and inserted into the pCS2 vector with indicated tags. Expression plasmids for caspase-1, 4, 5 and 11 were previously described4, 9, the caspase-9 plasmid was a gift from X. Wang (National Institute of Biological Sciences, Beijing). cDNAs for human CASP2 and mouse Casp8 are from the Life Technologies Ultimate ORF collection and OriGene (MC200404), respectively. Point mutations were generated by the QuickChange Site-Directed Mutagenesis Kit (Stratagene). All plasmids were verified by DNA sequencing. Antibodies for caspase-1 p10 (sc-515), Myc epitope (sc-789) and GSDMD (sc-81868) were obtained from Santa Cruz Biotechnology. Other antibodies used in this study include anti-HA (MMS-101P, Covance), anti-Flag M2 (F4049), anti-actin (A2066) and anti-tubulin (T5168) (Sigma-Aldrich), rat monoclonal caspase-11 17D9 (NB120-10454, Novus Biologicals), anti-caspase-3 (#9662) and caspase-7 (#12827) (Cell Signaling Technology), IL-1β (3ZD; Biological Resources Branch, National Cancer Institute) and the antibody for detecting endogenous GSDMD (NBP2-33422, Novus Biologicals). Ultrapure LPS from E. coli O111:B4 and poly(dA:dT) were purchased from InvivoGen. LPS (L4524, for priming), TNFα and cycloheximide were purchased from Sigma-Aldrich. SMAC mimetic and the pan-caspase inhibitor zVAD are gifts from the laboratory of X. Wang (National Institute of Biological Sciences, Beijing). Nigericin was purchased from Calbiochem. Recombinant p20/p10 active caspase proteins (caspase-1/2/4/8/9) and lipid A (ALX-581-200-L001) were obtained from Enzo Life Sciences. Cell culture products are from Life technologies and all other chemicals used are Sigma-Aldrich products unless noted. HeLa, HT-29 and 293T cells were obtained from ATCC. C57BL/6 mice-derived wild-type and Tlr4−/−iBMDM cells were kindly provided by K. A. Fitzgerald (University of Massachusetts Medical School, United States) and A. Ding (Weill Cornell Medical College, United States), respectively, and used in our previous studies4, 6, 30. All the cell lines are well-established, commonly used and frequently checked by virtue of their morphological features and functionalities, but have not been subjected to authentication by short tandem repeat (STR) profiling. All the cell lines have been tested to be mycoplasma-negative by the commonly used PCR method. iBMDM, HeLa and 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM); HT-29 cells were grown in McCoy’s 5a modified medium. All media were supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 2 mM l-glutamine. All cells were grown at 37 °C in a 5% CO incubator. Transient transfection of HeLa and 293T cells was performed using the JetPRIME (Polyplus Transfection) or Vigofect (Vigorous) reagents by following the manufacturers’ instructions. For stable expression, lentiviral plasmids harbouring the desired gene were first transfected into 293T cells together with the packing plasmids pSPAX2 and pMD2G with a ratio of 5:3:2. The supernatants were collected 48 h after transfection and used to infect HeLa or iBMDM cells for another 48 h. GFP-positive infected cells were sorted by flow cytometry (BD Biosciences FACSAria II). For siRNA knockdown, 0.5 μl of 20 μM siRNA together with 0.8 μl of INTERFERin reagents (Polyplus Transfection) were used for reverse transfection of iBMDM cells in the 96-well plate format; 5 μl of 20 μM siRNA and 10 μl of INTERFERin reagents were used to transfect HeLa cells in the 6-well plate format. The knockdown was performed for 60 h before subsequent analyses. The knockdown efficiency was assessed by quantitative real-time PCR (qRT–PCR) analyses as previously described4. All siRNA oligonucleotides were synthesized by our in-house facility using the sequences from the MISSION shRNA library (Broad Institute, United States) and their sequences are listed in Supplementary Table 1. Activation of the canonical caspase-1 inflammasomes (the NLRP3, NAIP–NLRC4 and AIM2 inflammasomes) and the non-canonical caspase-11 inflammasome by LPS was performed using the protocols that have been detailed in our previous publications4, 6, 9, 20. For bacteria-induced inflammasome activation, S. typhimurium (wild-type and ΔsipD), B. thailandensis (wild-type and ΔbipB), EPEC (wild-type and ΔescN) were used to infect iBMDM cells and S. typhimurium (wild-type and ΔsifA) was used to infect HeLa cells, as described previously4, 9. To examine cell death morphology, cells were treated as indicated in the 6-well plates (Nunc products, Thermo Fisher Scientific Inc.) for static image capture or in glass-bottom culture dishes (MatTek Corporation) for live imaging. Static bright field images of pyroptotic cells were captured using an Olympus IX71 or a Zeiss Pascal Confocal microscope. The image pictures were processed using ImageJ or the LSM Image Examiner program. Live images of cell death were recorded with the PerkinElmer UltraVIEW spinning disk confocal microscopy and processed in the software Volocity. All image data shown were representative of at least three randomly selected fields. The lentiviral gRNA plasmid library for genome-wide CRISPR-Cas9 screen was obtained from Addgene (#50947)33; amplification of the library and preparation of the lentivirus were performed following the protocol provided by Addgene. In brief, 1 μl of library DNA (10 ng μl−1) was used to transform 25 μl of electrocompetent E. coli (TaKaRa). Transformed colonies (>6 × 107) were scraped off the Luria-Bertani (LB) plates into the media, and plasmids were exacted by using the GoldHi EndoFree Plasmid Maxi Kit (CWBIO). To prepare the virus library, 293T cells in the 15-cm dish were transfected with 25 μg of library DNA together with 15 μg of psPAX2 and 10 μg of pMD2.G. Eight hours after transfection, the media were changed to high-serum DMEM (20% FBS with 25 mM HEPES). Another 40 h later, the media (from twenty 15-cm dishes of transfected cells) were collected and centrifuged at 3,000 r.p.m. for 10 min. The supernatant was filtered through a 0.22-μm membrane and aliquots of 30 ml were stored at −80 °C. In the pilot experiment, the volume of the lentivirus library required for achieving an MOI of 0.3 for infecting the target cell line was determined in the 12-well plate format. For the large scale screen, Tlr4−/− iBMDM cells stably expressing the Cas9 protein were seeded in the 15-cm dish (2 × 106 cells in 20 ml media per dish) and a total of 2 × 107 cells were infected with the gRNA lentivirus library. Sixty hours after infection, cells were re-seeded at a density of 1 × 105 ml−1 in fresh media supplemented with 5 μg ml−1puromycin (to eliminate non-infected cells). After 6 to 8 days, ~3 × 108 cells from five culture dishes were electroporated with LPS to trigger caspase-11-mediated pyroptosis9, or stimulated with LFn–BsaK/protective antigen (PA) to induce caspase-1-mediated pyroptosis4; another 3 × 108 cells were left untreated as the control sample. Each screen was repeated another time. Surviving cells were collected after growing to near 90% confluence and lysed in the SNET buffer (20 mM Tris-HCL(pH 8.0), 5 mM EDTA, 400 mM NaCl, 400 µg ml−1 Proteinase K and 1% SDS). Genomic DNAs of each group of cells were prepared by using the phenol-chloroform extraction and isopropanol precipitation method. The DNA was dissolved in H O (4–5 μg μl−1) and used as the templates for amplification of the gRNA. The gRNAs were amplified by a two-step PCR method using the Titanium Taq DNA polymerase (Clontech Laboratories). In the first step, six 50-μl PCR reactions (each containing 50 μg of genomic DNA template) were performed with the forward primer 50bp-F and the reverse primer 50bp-R; the PCR program used is 94 ° C for 180 s, 16 cycles of 94 ° C for 30 s, 60 ° C for 10 s and 72 ° C for 25 s, and a final 2-min extension at 68 ° C. Products of the first-step PCR were pooled together and used as the template for the second-step PCR. Also six 50-μl PCR reactions (each containing 1 μl of the first-step PCR product) were performed with the forward primer Index-F and one of the reverse primers (Index-R1 to R6): Index-R1 for the control sample, Index-R2 for the replicate control sample, Index-R3 for the caspase-11 screen, Index-R4 for the replicate caspase-11 screen, Index-R5 for the caspase-1 screen and Index-R6 for the replicate caspase-1 screen. The PCR program used is 94 ° C for 180 s, 18 cycles of 94 ° C for 30 s, 54 ° C for 10 s and 72 ° C for 18 s, and a final 2-min extension at 68 ° C. Products of the second-step PCR reactions were subjected to electrophoresis on the 1.5% agarose gel; the DNAs (the 310-bp band) were extracted and sequenced at the HiSeq2500 instrument (Illumina) by using the 50-bp single-end sequencing protocol. The first 19 nucleotides from each sequencing read are the gRNA sequence recovered from the library. The frequency of each gRNA was obtained by dividing the gRNA read number by the total sample read number; the fold of enrichment was calculated by comparing the frequency of each gRNA in the experiment sample with that in the control sample. Sequences for all the primers are listed in Supplementary Table 1. The top 50 gRNA hits from the caspase-11 screen were examined and 18 genes that are conserved in human and mouse were identified for siRNA knockdown validation in HeLa cells. HeLa cells expressed caspase-4 but not caspase-5 (Extended Data Fig. 1b) and respond robustly to cytosolic LPS9, 10. For each gene, a mixture of two independent siRNAs was used and the knockdown efficiency of 12 of those having mRNA expression in HeLa cells was confirmed. Importantly, only siRNAs targeting human GSDMD, besides the control CASP4-targeting siRNA, could efficiently block cytosolic LPS-induced pyroptosis (Extended Data Fig. 1c). When assayed individually, the two GSDMD-targeting siRNAs both showed potent inhibition of HeLa cell pyroptosis (Extended Data Fig. 1d). Human codon-optimized Cas9 (hCas9) and GFP-targeting gRNA-expressing plasmids (gRNA_GFP-T1) were purchased from Addgene. The 19-bp GFP-targeting sequence in the gRNA vector was replaced with the sequence targeting the desired gene by QuickChange site-directed mutagenesis. The target sequences used are AGCATCCTGGCATTCCGAG for mouse Gsdmd and TTCCACTTCTACGATGCCA for human GSDMD. To construct the knockout cell lines, 1 μg of gRNA-expressing plasmid, 3 μg of hCas9 plasmid and 1 μg of pEGFP-C1 vector were co-transfected into 6 × 106 iBMDM or HeLa cells. Three days later, GFP-positive cells were sorted into single clones into the 96-well plate by flow cytometry using the BD Biosciences FACSAria II or the Beckman Coulter MoFlo XDP cell sorter. Single clones were screened by the T7 endonuclease I-cutting assay and the candidate knockout clones were verified by sequencing of the PCR fragments as described previously9. The PCR primers used are listed in Supplementary Table 1. All animal experiments were conducted following the Ministry of Health national guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee at National Institute of Biological Sciences. The Gsdmd knockout mice were generated by co-microinjection of in vitro-translated Cas9 mRNA and gRNA into the C57BL/6 zygotes. Founders with frameshift mutations were screened with T7E1 assay and validated by DNA sequencing. Founders were intercrossed to generate biallelic Gsdmd−/− mice. The gRNA sequence used to generate the knockout mice is AGCATCCTGGCATTCCGAG. C57BL/6 wild-type mice were from Vital River Laboratory Animal Technology Co. and Casp1/11−/− mice were obtained from the Jackson Laboratory. Ripk3−/− mice were a gift from X. Wang (National Institute of Biological Sciences, Beijing). Primary BMDM cells were prepared from 6-week-old male mice (C57BL/6 background) by following a standard procedure as previously described6. For each experimental design, at least two mice were chosen to prepare the BMDM cells for assaying the inflammasome responses; the mice were not randomized and the investigators were not blinded. Relevant cells were treated as indicated. Cell death was measured by the LDH assay using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega). To measure IL-1β release, primary BMDM cells were primed with LPS (1 μg ml−1) for 2 h and released mature IL-1β was determined by using the IL-1β ELISA kit (Neobioscience Technology Company). To obtain recombinant human GSDMD, E. coli BL21 (DE3) cells harbouring pET28a-His -SUMO-GSDMD were grown in LB medium supplemented with 30 μg ml−1 kanamycin. Protein expression was induced overnight at 18 ° C with 0.4 mM isopropyl-B-d-thiogalactopyranoside (IPTG) after OD reached 0.8. Cells were harvested and resuspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 20 mM imidazole and 10 mM 2-mercaptoethanol. The His -SUMO-tagged protein was first purified by affinity chromatography using Ni-NTA beads (Qiagen) and the SUMO tag was removed by overnight ULP1 protease digestion at 4 °C. The cleaved GSDMD was further purified by HiTrap Q ion-exchange and Superdex G200 gel-filtration chromatography (GE Healthcare Life Sciences). To obtain the constitutive-active caspase-11 p20/p10 tetramer, cDNAs encoding the p20 large and p10 small subunit were cloned into pET21a with a 6×His tag fused to the C terminus of the p10 subunit. The two subunits were separately expressed in E. coli with 1 mM IPTG induction for 4 h at 30 ° C. Bacteria collected from 1-l culture were resuspended and lysed in 100 ml of lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol) by sonication. Inclusion bodies, obtained by centrifugation of the lysates at 18,000 r.p.m. for 1 h, was washed with 50 ml of Buffer 1 (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 M guanidinium hydrochloride (GdnCl) and 0.1% Triton X-100) and 50 ml of Buffer 2 (50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 1 M GdnCl) twice for each buffer. The washed inclusion bodies were solubilized by stirring in 6 ml of the solubilization buffer containing 6.5 M GdnCl, 25 mM Tris-HCl (pH 7.5), 5 mM EDTA and 100 mM DTT overnight at room temperature. To obtain active p20/p10 tetramers by refolding, 12 ml of above solubilized inclusion body solution containing denatured large and small subunits (molecular ratio, 1:2) were drop-by-drop diluted in 500 ml of refolding buffer (100 mM HEPES, 100 mM NaCl, 100 mM sodium malonate, 20% sucrose, 0.1 M NDSB-201 and 10 mM DTT) and then gently stirred in a nitrogen atmosphere at 16 °C overnight. Protein aggregates were removed by centrifugation at 4,000 r.p.m. for 20 min and the refolded protein supernatants were concentrated and dialysed against a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol. The protein was affinity-purified by the Ni-NTA beads and further purified by the Superdex G200 gel-filtration chromatography. Expression and purification of recombinant LFn–BsaK and LFn–FliC proteins were described previously4. Recombinant full-length caspase-4 and caspase-11 were expressed and purified from insect cells also as previously described9. Recombinant PreScission protease (PPase) proteins are routine lab stocks. For cleavage by the p20/p10 tetramers of active caspase, 5 μg of purified recombinant GSDMD was incubated with 1 unit of caspase-1, 2, 4, 8 and 9 or 0.1 μg of caspase-11 in a 25-μl reaction containing 50 mM HEPES (pH 7.5), 3 mM EDTA, 150 mM NaCl, 0.005% (vol/vol) Tween-20 and 10 mM DTT. The reaction was incubated for 60 min at 37 ° C. For cleavage by LPS-activated caspase-4/11, the full-length caspase proteins purified from insect cells were first incubated with LPS, lipid A or MDP for 30 min at 30 ° C; 5 μg of purified recombinant GSDMD was then reacted with the ligand-incubated caspases at 37 ° C for 9 min. Cleavage of GSDMD was examined by Coomassie blue staining of the reaction samples separated on the SDS–PAGE gel.
News Article | October 5, 2016
HEK293T cells23, commonly used in complex I assembly studies15, 20, 21, 24, 25, interactome26 and mitochondrial complexome studies24, were originally purchased from the ATCC and a clonal cell line was obtained after single cell sorting20 and used as the parental line for all gene editing and proteomic work. Knockout cell lines were validated by sequencing of targeted alleles for insertions and deletions (indels), immunoblotting and subsequent proteomic analysis. Cell lines regularly undergo testing for mycoplasma contamination using PlasmaTest (InvivoGen). Gene editing was performed using TALEN27 pairs as described15, 28, or the pSpCas9(BB)-2A-GFP (PX458) CRISPR/Cas9 construct (a gift from F. Zhang; Addgene, plasmid 48138; ref. 29). In brief, in the first round, TALEN constructs were designed using the ZiFiT Targeter30. For genes unsuccessfully targeted in the first round, CRISPR/Cas9 guide RNAs were designed for a second round of gene-disruption using CHOPCHOP31. Successful targeting strategies and constructs can be found in Supplementary Table 1. Gene edited and control HEK293T cells15 were cultured in DMEM (ThermoFisher) supplemented with 10% (v/v) FBS and 50 μg ml−1 uridine. Transfection reagents used were Lipofectamine 2000 and Lipofectamine LTX (ThermoFisher). During screening, glucose-free DMEM supplemented with 5 mM galactose, 1 mM sodium pyruvate, 10% (v/v) dialysed FBS (ThermoFisher) and 50 μg ml−1 uridine was used to identify respiratory incompetent knockout clones. Respiratory competent knockout clones were identified by sequencing of a mixed PCR product covering the target region, where a loss of sequencing fidelity at the target indicates a candidate clone28. With the exception of the NDUFA9- and COA6-knockout cell lines, which were described previously15, 20, indels for individual alleles are summarized in Supplementary Table 1. To generate NDUFAB1 knockout cells, clonal HEK293T cells were transduced with lentiviruses pLVX-TetOne-Puro-NDUFAB1*Flag or pLVX-TetOne-Puro-yACP1Flag (Clontech). NDUFAB1*Flag represents the C-terminally Flag-tagged human NDUFAB1 protein encoded by cDNA having undergone silent mutagenesis to remove the CRISPR/Cas9 target site. yACP1Flag indicates cDNA encoding the C-terminally Flag-tagged yeast (Saccharomyces cerevisiae) ACP1. Transduced cells were grown in the presence of 2 μg ml−1 puromycin for 72 h, and expression of NDUFAB1*Flag or yACP1Flag was confirmed after a further 72 h of treatment with 1 μg ml−1 doxycycline (DOX; Sigma-Aldrich) followed by SDS–PAGE and immunoblotting with NDUFAB1 (Abcam) and Flag (Sigma-Aldrich) antibodies. For subsequent gene editing, cells cultured in the presence of 50 ng ml−1 DOX were transfected with pSpCas9(BB)-2A-GFP-NDUFAB1 and screened as described above. For complementation, cDNAs encoding NDUFV3Flag, NDUFS6Flag, NDUFA8Flag, ATP5SLFlag and DMAC1Flag (TMEM261Flag) were cloned into pBABE-puro (Addgene, 1764; ref. 32), whereas NDUFA1, NDUFA2, NDUFB7, NDUFB10, NDUFB11 and NDUFC1 cDNAs were cloned into pBMN-Z (Addgene, 1734) in place of the LacZ insert. Retroviral constructs were used to transduce the corresponding main clone (Supplementary Table 1), following which expression was selected for through growth in galactose DMEM with the exception of NDUFS6 and NDUFV3 knockouts which were selected using 2 μg ml−1 puromycin. Transduction was verified by BN–PAGE or SDS–PAGE followed by immunoblotting with NDUFA9 or Flag antibodies, respectively. Mitochondria were isolated as previously described33. Protein concentration was estimated by bicinchoninic acid assay (BCA; Pierce), and aliquots of crude mitochondria stored at −80 °C until use. SDS–PAGE was performed using samples solubilized in LDS sample buffer and separated on NuPAGE Novex Bis-Tris protein gels according to manufacturer’s instructions (ThermoFisher). Tris-Tricine SDS–PAGE, BN–PAGE and 2D–PAGE were performed as described previously34, 35, 36. Carbonate and swelling experiments were performed as described37. Immunoblotting onto PVDF membranes was performed using a Novex Semi-Dry Blotter (ThermoFisher) according to manufacturer’s instructions. Horseradish peroxidase coupled secondary antibodies and ECL chemiluminescent substrate (BioRad) were used for detection on a BioRad ChemiDoc XRS+ imaging system. The following primary antibodies were used in this study: COX2 (ThermoFisher A-6404), COX4 (Abcam, ab110261), Flag (Sigma-Aldrich, M2 clone), MIC10 (Aviva Systems Biology, ARP44801_P050), NDUFA13 (Mitosciences MS103-SP), NDUFAB1 (Abcam, ab96230), NDUFB11 (Abcam, ab183716), NDUFV1 (Proteintech 11238-1-AP), NDUFS2 (Mitosciences, MS114), anti-respiratory-chain (Abcam, ab110413; which contains antibodies against ATP5A, UQCRC2, COX1, SDHB and NDUFB8), SDHA (Abcam, ab14715), TIMMDC1 (Sigma, HPA053214), TOMM20 (Santa Cruz, Sc11415) and UQCRC1 (ThermoFisher, 16D10AD9AH5), while rabbit polyclonal antibodies against NDUFA9 (ref. 12), NDUFAF1 (also known as CIA30)38, NDUFAF2 (ref. 21), NDUFAF4 (ref. 21), NDUFB6 (ref. 38) and HSP70 (ref. 20) were raised in-house. For analysis of mRNA expression levels, total RNA was obtained from each cell line in replicate with TRIzol (Thermo scientific). Total RNA was purified using Direct-zol columns according to the manufacturer’s specifications (Zymo Research). For cDNA synthesis, 1 μg of total RNA was processed as the T12VN-PAT assay39 adapted for multiplexing on the Illumina MiSeq instrument. We refer to this assay as mPAT for multiplexed PAT. The approach is based on a nested PCR that sequentially incorporates the Illumina platform’s flow-cell-specific terminal extensions onto 3′ RACE PCR amplicons. First, cDNA was generated using the anchor primer mPAT Reverse, next this primer and a pool of 50 gene-specific primers were used in 5 cycles of amplification. Each gene-specific primer had a universal 5′ extension (see Supplementary Table 12) for sequential addition of the 5′ (P5) Illumina elements. These amplicons were then purified using NucleoSpin columns (Macherey-Nagel), and entered into second round of amplification using the universal Illumina Rd1 sequencing Primer and TruSeq indexed reverse primers from Illumina. Second-round amplification was for 14 cycles. Note, that each experimental condition was amplified separately in the first round with identical primers. In the second round, a different indexing primer was used for each experimental condition. All PCR reactions were pooled and run using the MiSeq Reagent Kit v2 with 300 cycles (that is, 300 bases of sequencing) according to the manufacturer’s specifications. Data were analysed using established bioinformatics pipelines40. Figures were generated using the R framework. Oxygen consumption (OCR) and extracellular acidification (ECAR) rates were measured in live cells using a Seahorse Bioscience XF24-3 Analyzer as described15. In brief, 50,000 cells were plated per well in Seahorse Bioscience culture plates treated with 50 μg ml poly-d-lysine and grown overnight in standard culture conditions. The cellular OCR and ECAR were analysed in non-buffered DMEM (Seahorse Biosciences) containing 5 mM glucose, 1 mM sodium pyruvate and 50 μg ml−1 uridine with the following inhibitors: 2 μM oligomycin; 0.5 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP); 0.5 μM rotenone; and 0.3 μM antimycin A. For each assay cycle, four measurement time points of 2 min mix, 2 min wait and 5 min measure were collected. For each cell line, 3–4 replicate wells were measured in multiple plates and CyQuant (ThermoFisher Scientific) was used to normalize measurements to cell number. Basal OCR and non-mitochondrial respiration (following rotenone and antimycin A injections) were calculated as a mean of the measurement points. Basal ECAR was calculated from the initial basal measurement cycle. To calculate proton-leak and maximal respiration, the initial measurement following addition of oligomycin or FCCP was used. Enzymatic activity measurements were performed as previously described41 in three separate subcultures of each cell line. To accommodate unequal variance, statistical significance was determined through an unpaired two-sample, two-sided t-test using Welch’s correction. Radiolabelling of mtDNA-encoded proteins was performed as previously described15, 34. Isolated mitochondria were subjected to BN–PAGE or 2D–PAGE as described above, following which proteins were transferred to PVDF membranes and analysed by phosphorimager digital autoradiography (GE Healthcare Life Sciences). For immunoprecipitation of newly translated proteins, mitochondria were isolated from cells pulsed for 2 h and solubilized in 1% (w/v) digitonin, 20 mM Bis-Tris (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 10% (v/v) glycerol. After a brief clarification spin, complexes were incubated with anti-Flag affinity gel (SigmaAldrich), the gel washed with 0.2% (w/v) digitonin, 20 mM Bis-Tris (pH 7.0), 60 mM NaCl, 0.5 mM EDTA, 10% (v/v) glycerol, and enriched proteins eluted with the addition of 150 μg ml−1 Flag peptide (SigmaAldrich). Samples were TCA precipitated to remove detergent and analysed by SDS–PAGE and phosphorimaging as above. For protein import, NDUFA12, NDUFA7 and NDUFV3 cDNA was cloned into the pGEM-4Z plasmid (Promega). mRNA was transcribed using the mMESSAGE mMACHINE SP6 transcription kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Radiolabelled proteins were translated in the presence of [35S]methionine/cysteine using a rabbit reticulocyte lysate system (Promega). Translated proteins were incubated with isolated mitochondria at 37 °C as previously described12, following which mitochondria were analysed by SDS–PAGE or BN–PAGE as described above. For NDUFV3, NDUFS6, NDUFA2, NDUFA8, NDUFA1, NDUFS5, NDUFC1, NDUFB4, NDUFB7, NDUFB10 and NDUFB11 knockouts, mass spectrometry was performed with SILAC-labelled whole-cell starting material as described previously42 with modifications. In brief, cells cultured in ‘heavy’ 13C 15N -arginine, 13C 15N -lysine-containing or ‘light’ SILAC DMEM15 were collected, washed in PBS and protein content determined by BCA assay. Measurements were performed in batches of 3–4 knockout cell lines in triplicate with a label switch. Each batch used a single pool of clonal HEK293T cells (1 sample grown in heavy DMEM, and 2 independent samples grown in light DMEM) and knockout cell lines were grown with the complementary label orientation (1 in light DMEM, and 2 in heavy DMEM). Equal amounts of heavy and light (typically 250 μg) control HEK293T and knockout cells were mixed, and cells were solubilized in 1% (w/v) sodium deoxycholate, 100 mM Tris-HCl (pH 8.1). Lysates were sonicated for 30 min at 60 °C in a sonicator waterbath, followed by denaturation and alkylation through the addition of 5 mM Tris(2-carboxyethy)phosphine (TCEP), 20 mM chloroacetamide and incubation for 5 min at 99 °C with vortexing. Samples were digested with trypsin overnight at 37 °C. Detergent was removed by ethyl acetate extraction in the presence of 2% formic acid (FA), following which the aqueous phase was concentrated by vacuum centrifugation. Peptides were reconstituted in 0.5% FA and loaded onto pre-equilibrated small cation exchange (Empore Cation Exchange-SR, Supelco Analytical), stage-tips made in-house. Tips were washed with 6 load volumes of 20% acetonitrile (ACN), 0.5% FA and eluted in 5 sequential fractions of increasing amounts (45-300 mM) of ammonium acetate, 20% ACN, 0.5% FA. A sixth elution was collected using 5% ammonium hydroxide, 80% ACN following which fractions were concentrated, desalted and reconstituted as previously described15. Peptides were reconstituted in 0.1% trifluoroacetic acid (TFA) and 2% ACN and fractions analysed sequentially by online nano-HPLC/electrospray ionization-MS/MS on a Q Exactive Plus connected to an Ultimate 3000 HPLC (Thermo-Fisher Scientific). Peptides were first loaded onto a trap column (Acclaim C PepMap nano Trap × 2 cm, 100-μm I.D, 5-μm particle size and 300-Å pore size; ThermoFisher Scientific) at 15 μl min−1 for 3 min before switching the pre-column in line with the analytical column (Acclaim RSLC C PepMap Acclaim RSLC nanocolumn 75 μm × 50 cm, PepMap100 C , 3-μm particle size 100-Å pore size; ThermoFisher Scientific). The separation of peptides was performed at 250 nl min−1 using a nonlinear ACN gradient of buffer A (0.1% FA, 2% ACN) and buffer B (0.1% FA, 80% ACN), starting at 2.5% buffer B to 35.4% followed by ramp to 99% over 120 min (runs had a total acquisition time of 155 min to accommodate void and equilibration volumes). Data were collected in positive mode using Data Dependent Acquisition using m/z 375–1800 as MS scan range, HCD for MS/MS of the 12 most intense ions with z ≥ 2. Other instrument parameters were: MS1 scan at 70,000 resolution (at 200 m/z), MS maximum injection time 50 ms, AGC target 3E6, Normalized collision energy was at 27% energy, Isolation window of 1.8 Da, MS/MS resolution 17,500, MS/MS AGC target of 1E5, MS/MS maximum injection time 100 ms, minimum intensity was set at 1E3 and dynamic exclusion was set to 15 s. For the remaining knockouts, we used isolated mitochondria as starting material. Cells were cultured in SILAC DMEM as above and mitochondrial isolations performed in batches of 1–6 knockout cell lines in triplicate. Each batch contained a single set of clonal HEK293T mitochondria (2 independent isolations from heavy and 1 from light cells), with knockout mitochondria having the complementary label orientation (2 independent isolations from light DMEM and 1 from heavy cells). Mitochondria were isolated from cell pellets stored at −80 °C as previously described43, but with modifications. Cells were resuspended in 20 mM HEPES-KOH (pH 7.6), 220 mM mannitol, 60 mM sucrose, 1 mM EDTA, 1 mM PMSF and homogenized as described above. The homogenate was centrifuged at 800g for 5 min at 4°C, and the supernatant again centrifuged at 10,000g for 10 min at 4 °C. Crude mitochondria were resuspended in the above buffer and the two differential centrifugation steps repeated. The resuspended pellet was then layered onto a sucrose cushion consisting of 10 mM HEPES-KOH (pH 7.6), 500 mM sucrose, 1 mM EDTA. Samples were centrifuged at 10,000g for 10 min at 4 °C, following which the protein concentration was estimated by BCA assay. Equal amounts of heavy and light (typically 20 μg) control HEK293T and knockout mitochondria were mixed as described above, collected by centrifugation at 18,000g and solubilized in 8 M urea, 50 mM ammonium bicarbonate. Proteins were acetone-precipitated, reduced and alkylated and desalted as previously described15. Peptides reconstituted in 0.1% TFA and 2% ACN were analysed on a Q Exactive Plus, or a LTQ-Orbitrap Elite Instrument. Instrument and method parameters for Q Exactive Plus were as described above, however, used a shorter gradient (90 min separation, 120 min total acquisition). For the Orbitrap Elite, instrument and method parameters were as previously described15. A single technical re-injection was collected for all mitochondrial samples. All raw file names included identifiers for the batch, instrument and gradient used, knockout cell line being studied, and applicable label orientation. Raw files were analysed using the MaxQuant platform44 version 18.104.22.168, searching against the Uniprot human database containing reviewed, canonical and isoform variants in FASTA format (June 2015) and a database containing common contaminants by the Andromeda search engine45. Default search parameters for an Arg10- and Lys8-labelled experiment were used with modifications. In brief, cysteine carbamidomethylation was used as a fixed modification, and N-terminal acetylation and methionine oxidation were used as variable modifications. False discovery rates of 1% for proteins and peptides were applied by searching a reverse database, and ‘re-quantify’ and ‘match from and to’, ‘match between runs’ options were enabled with a match time window of 2 min. Experimental groups based on data gathered using different instrumentation and/or acquisition parameters were given odd numbered fractions to avoid falsely matched identifications, whereas fractionated whole-cell samples were given sequential fraction numbers. Unique and razor peptides with a minimum ratio count of 2 were used for quantification. Using the Perseus platform (version 22.214.171.124), identifications were matched to the MitoCarta2.0 database19 using Ensembl ENSG id and gene name identifiers. Identifications labelled by MaxQuant as ‘only identified by site’, ‘reverse’ and ‘potential contaminant’ were removed. Proteins having <3 valid values in a single experimental group were removed. For mitochondrial samples, we found the correlation of log -ratio data from biological replicates in the same experimental group to be moderate at best and as low as 0.3 in some cases. We surmised the main cause of this to be batch and labelling effect, the former due to differences in mitochondrial isolations between batches and latter due to one (of three) replicates within each experimental group always being subjected to a label switch. To account for these and potentially other factors, we adopted an approach that borrows principles from RUV-2 (ref. 46) and SVA47 methods for removing unwanted variations, with modifications in the algorithm for choosing the control proteins (that is, those not found in MitoCarta 2.0; ref. 19) and moderating the amount of adjustment for genes with small sample size due to missing values. Adjustments were performed in the R framework, following which the adjusted ratios were imported back into Perseus. The log ratio values for proteins in replicates were normally distributed and had equal variances. The mean log -transformed ratios for each experimental group were calculated along with their standard deviation and P-value based on single sample two-sided t-test15. This statistical approach was consistent with published quantitative SILAC analyses employing similar instrumentation and methods15, 48, 49. Groups having <2 valid values were converted to ‘NaN’ (not a number). A quality matrix was generated based on the standard deviation, and corresponding values having a standard deviation greater than 1 converted to ‘NaN’. This threshold was determined empirically to remove outliers from the main distribution of standard deviations across all samples. These data can be found in Supplementary Table 5. Figures 3b and Extended Data Figs 6a, 8c and 9d were generated from a matrix containing log -transformed median SILAC ratios having a standard deviation <1 for complex I subunits (Supplementary Table 7) and data were mapped to homologous subunits (Protein Data Bank accession 5LDW)9. For Fig. 3a, hierarchical clustering on rows (proteins) was performed using Pearson distance and average linkage. Data were pre-processed using k-means (clusters = 300). Images were generated using the PyMOL Molecular Graphics System, version 126.96.36.199 (Schrödinger, LLC). log SILAC ratios for some proteins in their corresponding knockout cell line had very low (generally >4-fold reduction) ratios, whereas others were reported NaN. This could be either due to the ‘re-quantify’ option being turned on for the MaxQuant search, which results in translation of peak shapes from an identified isotope pattern being translated to its unidentified label partner, or indels in some lines generating a non-functional (but still translated) protein as we have seen previously15. For the identification of proteins dysregulated between knockouts of discrete modules (Fig. 4c, Supplementary Tables 8 and 9), triplicate log -transformed SILAC ratios from Supplementary Table 5 were assigned to one of two groups based on the knockout being associated with the indicated module. Groups tested had comparable variance, and a modified Welch’s two-sample t-test with permutation-based FDR statistics50, 51 was used to determine significance. Parameters for the test were: 70% minimum valid values, 250 permutations and significance being an FDR of <0.05. For the Gene Ontology enrichment analysis in Fig. 2c, proteins with a P < 0.05 and with >1.5-fold change up or down were submitted to the DAVID online tool (https://david.abcc.ncifcrf.gov/home.jsp) for enriched biological processes (GOTERM_BP_FAT) and molecular function (GOTERM_MF_FAT). Functional annotation charts were exported and visualized using Cytoscape (version 3.4.0) and the Enrichment Map app52 (version 2.1.0; P < 0.005). Contents of enriched terms indicated in Fig. 2c are detailed in Extended Data Fig. 5d. Affinity-enrichment experiments in Fig. 4e, Extended Data Figs 3d and 4d and Supplementary Tables 2–4 and 10, 11 were performed from HEK293T and knockout cells complemented with the Flag-tagged protein cultured in heavy or light SILAC DMEM as previously described15. Mass spectrometry was performed on a Q Exactive Plus as above but using a shorter gradient (25 min separation, 60 min total acquisition). For data analysis, raw files were analysed using the MaxQuant platform as above using default search parameters for a Arg10 and Lys8 labelled experiment, with modifications. In brief, cysteine carbamidomethylation was used as a fixed modification, and N-terminal acetylation and methionine oxidation were used as variable modifications. False discovery rates of 1% for proteins and peptides were applied by searching a reverse database, and ‘re-quantify’ and ‘match from and to’, ‘match between runs’ options were enabled with a match time window of 2 min. Unique and razor peptides with a minimum ratio count of 1 were used for quantification. Data analysis was performed using the Perseus framework. Identifications were matched to MitoCarta2.0 data set19 as above. Only proteins with a sequence coverage of 2 or more unique peptides were included in further analysis. Normalized SILAC ratios were inverted to achieve the orientation Flag-tagged/HEK293T and proteins not present in >2/3 replicates were removed. log -transformed values had a normal distribution and comparable variance. For affinity-enrichment experiments, statistical method, sample size and analysis approaches were chosen based on published quantitative affinity-enrichment analyses employing similar instrumentation and methods15, 21, 53, 54. P values were calculated by a single (Flag-tagged cell line enriched)-sided t-test and the negative logarithmic P-value plotted against the mean of the three replicates. cDNA inserts were obtained from an in-house cDNA library generated from our clonal HEK293T line. Briefly, RNA was isolated using TRIzol Reagent (ThermoFisher) according to manufacturer’s instructions. The Superscript III first strand synthesis kit (ThermoFisher Scientific) was used to generate cDNA primed with either Oligo(dT) or random hexamers. Inserts were amplified from the library using Q5 High Fidelity DNA Polymerase (NEB) and Gibson assembled into the relevant plasmid (see above) using the NEBuilder HiFi DNA Assembly Master Mix (NEB) according to manufacturer’s instructions. Sanger sequencing was performed from PCR product or plasmid template DNA. DNA sequence assembly and alignment to sequencing reads was performed using SnapGene (GSL Biotech) and Geneious (Biomatters). Immunofluorescence microscopy was performed as previously described55 using primary antibodies (Flag or TOMM20) at 1:500 dilutions. Primary antibodies were labelled with anti-mouse conjugated Alexa Fluor 488 and anti-rabbit conjugated Alexa Fluor 568 secondary antibodies (Molecular Probes). Hoechst (1 μg ml−1) was used to stain nuclei. Cells were visualized using a Leica TCS SP8 equipped with HyD detectors. Images were processed using Image J56. All figures were prepared using Adobe Photoshop and Illustrator (CC2015.5). 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.
News Article | October 25, 2016
Plvaptm1Salm (Plvap−/−), Nt5etm1Lft (Nt5e−/−) and Aoc3tm1Salm (Aoc3−/−) mice have been previously described24, 31, 32. Nt5e−/− and Aoc3−/− mice manifest with altered leukocyte trafficking33. We produced inducible PlvapF/F; CAGGCre-ERTM mice by crossing conditional PlvapF/F mice with a CAGGCre-ERTM (B6.Cg-Tg(CAG-cre/Esr1)5Amc/J (stock 004682 from the Jackson Laboratory) deletor Cre mouse line, in which Cre-ER is ubiquitously expressed after tamoxifen injection34. To achieve selective deletion of Plvap during embryonic development in LYVE-1+ endothelial cells, including yolk-sac endothelium and liver sinusoidal endothelial cells, but not the majority of other blood vessels35, conditional PlvapF/F mice were crossed with Lyve1-Cre mice (Lyve1tm1.1(EGFP/cre)Cys, stock 012601 from the Jackson Laboratory36) to generate PlvapF/F; Lyve1-Cre mice. Cav1tm1Mls/J (Cav1−/−, stock 004585) mice, which lack all caveolae37, were obtained from the Jackson Laboratory. C57BL/6J, C57BL/6N and BALB/c mice were purchased from Charles River and Janvier labs. F1 hybrid C57BL/6;129 (stock 101045) mouse strain was obtained from the Jackson Laboratory. Both genders were used in the experiments (except in mammary gland analyses). Sex-matched wild-type littermate mice were used as controls in each experiment. Embryonic development was estimated considering the day of vaginal plug as embryonic age of 0.5 days (E0.5). The adult mice were 4–5 weeks old, since few Plvap−/− mice survive till early adulthood24, 28, 38. All animal experiments were approved by the Ethical Committee for Animal Experimentation in Finland. They were carried out in adherence with the rules and regulations of the Finnish Act on Animal Experimentation (497/2013) and in accordance to the 3R-principle under Animal License number 5587/04.10.07/2014. Genotyping of Plvap−/−, Cav1−/−, Nt5e−/− and Aoc3−/− mice was performed according to protocols described previously24, 31, 32, 37. PlvapF/F; CAGGCre-ERTM and PlvapF/F; Lyve1-Cre mice genotyping was conducted using the following primers: primers A (3′-GTACATGCAACACCACTGAGC-5′) and B (3′-CCTTGACAGGTGATGTCTGC-5′) detect the wild-type Plvap allele (a 210-bp fragment) and the targeted Plvap allele (a 310-bp fragment; data not shown). Genotyping of CAGGCre-ERtm and Lyve1-Cre was done according to protocols described previously34, 36. Pregnant females were killed by carbon dioxide inhalation and cervical dislocation. Embryos from E10.5–E16.5 were dissected out from uterus and immersed in cold PBS (Invitrogen). The blood was collected after decapitation to heparin-containing tubes. Liver, lungs, spleen and brains were carefully dissected from the embryo and the yolk sac was collected. To obtain single-cell suspensions, the organs were incubated in Hank’s buffered saline (HBS) containing 1 mg ml−1 collagenase D (Roche), 50 μg ml−1 DNase I (Sigma) at 37 °C in 5% CO (30 min for liver, lung, spleen and brain and 2 h for yolk sac), and then passed through a 70-μm cell strainer. Erythrocytes were lysed from the blood and spleen samples as described24. The brain cells were re-suspended in isotonic Percoll and the microglia were isolated as described6. The cells from the adult tissues were isolated by the same method with some modifications. The blood was collected by a cardiac puncture into heparinized tubes. Lymph nodes, lung tissue and mammary fat pads were mechanically dissociated before a 60-min collagenase D and DNase I digestion. Livers were dissociated using Gentle MACS C-tube (Miltenyi Biotech) and immune cells were purified via OptiPrep density gradient centrifugation (Sigma D1556). The bone marrow was isolated by gently crushing the femurs before filtration. Lamina propria cells from the colon were isolated by an enzymatic digestion as described23. Peritoneal cells were collected by flushing the peritoneal cavity with RPMI 1640 supplemented with 2% FBS and 5 IU ml−1 heparin. Total leukocyte numbers in different organs were enumerated by determining the absolute numbers of viable cells in the cell suspensions by an automated cell counter (Cellometer Auto 2000, Nexcelcom) and the percentage of CD45+ cells (and of the various leukocyte subpopulations) by flow cytometry. Absolute leukocyte numbers in the blood were counted using an automated haemocytometer (VetScan HM5, Abaxis). Pregnant heterozygous Plvap−/+ females were transiently treated with anti-CSF-1R monoclonal antibody (clone AFS98, Bio X Cell) or with the rat IgG2a isotype control (clone 2A3, Bio X Cell) at E6.5 using a single intraperitoneal injection (3 mg of antibodies in sterile PBS). This treatment prevents the development of yolk sac macrophages, but does not affect EMP development10, 39. The fluorochrome-conjugated monoclonal antibodies (the antibody clones, fluorochromes, suppliers and catalogue numbers) against mouse molecules that were used for flow cytometry stains are listed in Supplementary Table 2. Before staining, the cell suspensions were incubated with purified anti-CD16/32 (clone 2.4G2, 553142 from Becton Dickinson) for 10 min on ice to block non-specific binding to Fc-receptors. Isotype-matched negative control antibodies conjugated to the appropriate fluorochromes were used (Supplementary Table 2). All FACS analyses were run using an LSRFortessa flow cytometer (BD Biosciences) and analysed using FlowJo (TreeStar) software. The FACS gates used to define each leukocyte subpopulation in different organs and tissues of embryos and adult mice are shown in Extended Data Figs 1, 2b, d, g, 4b, 5c–e, 10d and Supplementary Table 1. EMPs (CD41+CD45+c-KithighF4/80− cells) from E10.5 yolk sac, and yolk-sac-derived macrophages (CD11b+F4/80high cells) and fetal liver-derived macrophages (CD11b+F4/80intermediate cells) from E14.5 (wild-type and Plvap−/−) and E16.5 (wild-type) fetal livers (mechanical dissociation without enzymatic digestions) were sorted from embryos using Sony SH800Z (100-μm nozzle, Sony Biotechnology) and FACS aria II (70-μm nozzle, Becton Dickinson) cell sorters. The purity of the isolated populations was >95%. Approximately 5,000 macrophages sorted from fetal livers (see above) were spun down onto microscopic slides using a cytospin centrifuge (Shandon cytospin III, Tecan). The cells were stained with Diff-Quick (REASTAIN), and photographed using Zeiss AxioVert 200M (Zeiss) using a Plan-Noefluar 40×/0.60 objective. Fetal livers from E12.5–E16.5 embryos were excised from the mice after decapitation. They were embedded in optimal cutting temperature (OCT) compound and snap-frozen. Cryostat sections (6 μm in thickness) were cut and fixed in ice-cold acetone. The sections were overlaid with the following antibodies: Alexa Fluor 488-conjugated rat anti-mouse F4/80 (MF48020, Invitrogen), allophycocyanin-conjugated rat anti-mouse CD31 (102510, BioLegend), rat monoclonal anti-mouse MECA32 (550563, Becton Dickinson), rat anti-mouse MAdCAM-1 (MECA-367; rat IgG2a, a gift from E. Butcher), rabbit polyclonal anti-mouse LYVE-1 (102-PA50AG or 103-PA50; Reliatech), rabbit polyclonal anti-mouse caveolin (SC-894; Santa Cruz) and rabbit polyclonal anti-VEGF (46154, Abcam). Alexa Fluor 647-conjugated goat anti-rat immunoglobulin (A21247, Life Technologies), Alexa Fluor 546-conjugated goat anti-rabbit immunoglobulin (A11035 and A11035, highly cross-absorbed, Invitrogen), Alexa Fluor 633-conjugated goat anti-rabbit immunoglobulin (highly cross-adsorbed A21071, Life Technologies) and Alexa Fluor 488-conjugated donkey anti-rat immunoglobulin (highly cross-adsorbed, A21208, Life Technologies) were used as secondary antibodies as appropriate. The sections were mounted in ProLong Gold with or without DAPI (4′,6-diamidino-2-phenylindole). For visualization of the luminal location of PLVAP in fetal liver sinusoids at E12.5, wild-type C57BL/6N dams were killed at E12.5 and the embryos were excised from the uterine cavity but kept inside the yolk sac in warm PBS. Then 20 μg of unconjugated rat monoclonal anti-mouse MECA-32 (MECA32, custom-made, InVivo BioTech) or isotype-matched control antibody (rat IgG2a 553926 BD) was injected to umbilical and vitelline veins of the yolk sac. After 1 min, the embryos were decapitated, and the livers were collected and processed for ex vivo immunostaining. Acetone-fixed frozen sections were sequentially stained with Alexa Fluor 647-conjugated goat anti-rat immunoglobulin (A21247, Life Technologies) to detect the in vivo bound MECA-32, rabbit anti-mouse LYVE-1 (103-PA50, Reliatech) and Alexa Fluor 546-conjugated goat anti-rabbit immunoglobulin (A11035, Invitrogen). Preliminary analyses verified that species-specific second-stages antibodies showed no cross-reactivity with primary antibodies generated in the other species. Whole-mount immunohistochemistry from optically cleared E8.5 and E10.5 yolk sac and AGM, E9.5 embryos and E14.5 fetal livers was done as described previously24. Primary antibodies were rat monoclonal antibodies against mouse CD117 (c-Kit, 553352, Becton Dickinson), MECA-32 (PLVAP, 550563, Becton Dickinson), CD31 (550274, Becton Dickinson) and rabbit anti-mouse LYVE-1 (103-PA50, Reliatech). Alexa Fluor 488-conjugated donkey anti-rat immunoglobulin (A21208), Alexa Fluor 546-conjugated goat anti-rat immunoglobulin (A11081), Alexa Fluor 647-conjugated goat anti-rat immunoglobulin (A21247), and Alexa Fluor 633-conjugated goat anti-rabbit immunoglobulin (A21071) were used as secondary antibodies (all from Life Technologies). In AGM, c-Kit is expressed in HSCs, and CD31 in endothelial cells and HSCs40. A human fetal liver sample (pregnancy week 18) was cut, acetone-fixed and stained with monoclonal PAL-E (against human PLVAP; Ab8086, Abcam) and Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (highly cross-absorbed, A11029, Invitrogen). After staining, the sections were mounted in Prolong Gold with DAPI. Images were acquired with a LSM 780 confocal microsope (Zeiss) using a c-Apochromat 40×/1.20 W Korr M27 objective or plan-apochromat 20×/0.8 objective (Fig. 4a and Extended Data Fig. 4g) and Zen 2010 software (Zeiss). Using pinhole adjustments, a slice thickness of 4.6 μm and 1.2 μm was used for 20× and 40× objectives, respectively. A background subtraction was used for all images. In certain images, the brightness was linearly changed and noise was reduced using mean filter in ImageJ software. Brightness adjustments and noise reductions were always applied equally to images captured from wild-type and PLVAP-deficient mice. Splenic F4/80high cells were quantified by thresholding the images so that only the F4/80high cells remained visible (the thresholding was applied equally to images captured from wild-type and PLVAP-deficient mice). Thereafter, white pulp areas were excluded (based on MadCAM-1 staining in marginal zone41) and the area fraction in the red pulp containing the F4/80high cells was measured using ImageJ software. A spleen area of at least 2.1 mm2 per mouse was analysed. Z-stacks and images (Extended Data Fig. 4a E8.5 yolk sac) were acquired from the optically cleared samples using a 3i Spinning Disk confocal microscope (Intelligent Imaging Innovations) with a plan-apochromat 20×/0.8, 10×/0.45 or LD c-apochromat 40×/1.1 W objective. Background subtractions, linear brightness adjustments and mean filter noise reductions were done using ImageJ software. To produce maximum projections with SlideBook 6 software (Intelligent Imaging Innovations, Inc.), 15–87 sections with slice thickness of 0.63 μm were used (Extended Data Fig. 4a E10.5 yolk sac and 4g). 3D reconstruction was generated from Z-stacks (44 slices with thickness of 2.34 μm (MECA-32 in Supplementary Video 1), 78 slices with slice thickness of 2.34 μm (CD31 in Supplementary Video 1), and 117 slices with thickness of 0.43 μm (MECA-32 in Supplementary Video 2)) and converted to AVI file format with Imaris 8.0 software (Bitplane). Formalin-fixed, paraffin-embedded sections of livers from E11.5–E16.5 wild-type and Plvap−/− mice were cut, deparaffinized and subjected to heat-mediated antigen retrieval in EDTA buffer (Dako S2367). Endogenous peroxide was quenched with 3% H O , non-specific immunoglobulin binding was blocked with rabbit serum, and endogenous biotin and avidin were blocked using DakoCytomation Biotin blocking system (Dako, X0590). The liver sections were incubated overnight at 4 °C with the primary antibody (MECA-32, 1 μg ml−1 in PBS). A secondary antibody (biotinylated anti-rat immunoglobulin) was incubated for 30 min, and then biotin–avidin complexes were formed with using Vectastain ABC kit (PK-6100, Vector Laboratories). Liquid DAB+ substrate Chromogen System (Dako K3468) was used to oxidize and detect the peroxidase complexes. Finally, samples were stained with haematoxylin, dehydrated and mounted. Macrophage-dependent iron recycling was studied in spleen and liver by measuring accumulation of ferric ion42, 43. For Fe3+ stains, the spleens and livers from 5-week-old wild-type and Plvap−/− mice were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0), embedded in paraffin and sectioned. The detection of ferric iron was accomplished using the Prussian blue histological staining method, as described previously25, 43. The slides were counter-stained in nuclear Fast Red for 5 min. The slides were analysed using a Panoramic 250 Flash II slide-scanner (3D Histech). In spleen tissue, the white pulp areas were excluded and the red pulp areas containing the blue-stained Fe3+-containing cells were analysed using image thresholding. Area fractions were measured using ImageJ software. A spleen area of at least 1.5 mm2 per mouse was analysed. In livers, whole sections (at least 21.1 mm2 per mouse) were analysed. The fourth mammary gland was dissected from 4.5-week-old wild-type and Plvap−/− mice, and left to adhere to the object glass. The tissue was fixed by submerging in Carnoy’s medium (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 °C, and rehydrated in decreasing ethanol concentration series. The slides were then stained with carmine alum (0.2% carmine, 0.5% aluminium potassium sulfate dodecahydrate) overnight at room temperature, dehydrated, cleared in xylene for 2–3 days, and mounted in DPX Mountant (Sigma). The samples were imaged with Zeiss SteREO Lumar V12 stereo microscope using NeoLumar 0.8× objective and Zeiss AxioCam ICc3 colour camera. Several images were automatically combined into a mosaic picture using Adobe Photoshop. The area covered by the ductal tree, and the number of ductal branches was tracked manually and quantified using ImageJ with ‘Skeletonize2D/3D’ and ‘AnalyzeSkeleton’ plugins. Livers from E12.5, E14.5 and E16.5 wild-type and Plvap−/− embryos were collected and fixed in 5% glutaraldehyde in 0.16 M s-collidine buffer, pH 7.4. The samples were post-fixed for 2 h with 2% OsO containing 3% potassium ferrocyanide, dehydrated with a series of increasing ethanol concentrations (70%, 96% and twice at 100%) and embedded in 45359 Fluka Epoxy Embedding Medium kit. 70-nm sections were cut with an ultramicrotome, and stained with 1% uranyl acetate and 0.3% lead citrate. The sections were examined with a JEOL JEM-1400 Plus transmission electron microscope. After isolation, 5,000 cells from the yolk sac of E10.5, liver of E12.5 embryos and adult bone marrow of wild-type and Plvap−/− mice were seeded in 1 ml of M3434 Methocult medium (Stem Cell Technologies) into 35-mm culture dishes in duplicates. After a 7-day culture at 37 °C with 5% CO the number of colonies was counted, as described44. Total RNA was isolated from fetal livers of wild-type and PLVAP-deficient mice using the Nucleo-Spin RNA kit (Macherey–Nagel) and from the sorted EMP and macrophages (see above) using the RNAeasy Plus Micro kit (Qiagen). The RNA was reverse-transcribed to cDNA with SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific) according to the manufacturers’ instructions. Quantitative PCR (qPCR) was carried out using Taqman Gene Expression Assays (ThermoFisher Scientific) for Plvap (Mm00453379_m1; target gene), Lyve1 (Mm00475056_m1; target gene) and Actb (Mm00607939_s1; control gene). The expression of reported signature transcripts7 enriched in yolk-sac-derived F4/80high macrophages (Cx3cr1 (Mm00438354_m1), Mrc1 (Mm00485148_m1), Adgre1 (Mm00802529_m1, also known as Emr1 or F4/80), and in F4/80intermediate fetal liver-derived monocytes (Itgam (Mm00434455_m1), Gata2 (Mm00492301_m1), Flt3 (Mm00439016_m1) and Ccr2 (Mm04207877_m1)) in E16.5 fetal liver of wild-type mice was also analysed by quantitative qPCR. The reactions were run using the 7900HT Fast Real-Time PCR System (Applied Biosystems/ ThermoFisher Scientific) or QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems/ ThermoFisher Scientific) at the Finnish Microarray and Sequencing Centre (FMSC), Turku Centre for Biotechnology, Turku, Finland. Relative expression levels were calculated using Sequence Detection System (SDS) Software v2.4.1, QuantStudio 12 K Flex software, and DataAssist software (all from Applied Biosystems/ThermoFisher Scientific). The results were presented as a percentage of the control gene mRNA level from the same samples. A PLVAP–Fc fusion protein expressing the extracellular domain of mouse PLVAP fused to human IgG2 Fc-tail was generated (Extended Data Fig. 9c). The extracellular domain (amino-acid residues 48–438) was PCR-cloned from a full-length cDNA clone for mouse PLVAP (MR206983, Origene) using primers introducing EcoRV and NheI digestion sites. The PCR reaction was carried out using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific). The amplified fragment was purified and annealed to EcoRV and NheI digested pFUSEN-hG2Fc vector (InvivoGen) designed for the production of Fc-chimaeras from type 2 membrane proteins. The intactness of the construct was verified by sequencing, and its reactivity with anti-PLVAP antibody MECA-32 using immunoblotting. The expression plasmid was transfected into HEK293-EBNA cells (CRL-10852, from ATCC) using lipofection (Lipofectamine, Invitrogen), the cells were cultured for 2–3 days in serum-free medium (Pro293A-CDM, Bio-Whittaker). A CD4–Fc chimaera45 was used as a control. For heparin-affinity pull-down assays, agarose beads coupled to heparin (Sigma) or streptavidin (negative-control beads, from GE Healthcare) were washed, and blocked with TBS (pH 7.2) containing 1% BSA. The beads were rocked with clarified 0.5% NP-40 total protein lysates from E14.5 wild-type livers for 2 h at 4 °C in TBS containing 1% BSA. Alternatively, PLVAP–Fc and CD4–Fc fusion proteins were applied to the heparin and control beads. After washing with TBS containing 0.3% NP-40, the bead-bound molecules were eluted in Laemmli’s sample buffer, and subjected to SDS–PAGE separation. In certain experiments, the heparin and control beads were incubated with the fusion proteins and, after washing, the same volumes of the beads were eluted directly in Laemmli’s sample buffer or in 1.0 M NaCl, and the eluted proteins from the supernatants were submitted to SDS–PAGE. In other specificity control experiments, the binding of PLVAP–Fc fusion protein to heparin-beads was analysed in the absence and presence of 100 μg of fibronectin (F1141, Sigma), which binds to heparin46, or 100 μg of collagen (C8919, Sigma). After transfer to nitrocellulose membranes, the bound molecules were visualized using immunoblotting with a horseradish-peroxidase-conjugated anti-human IgG antibody (81-7120, Invitrogen; for the chimaeras) or with anti-PLVAP (MECA-32, BioXCell), anti-neuropilin-1 (AF56615, R&D Systems), and anti-VEGF (sc-152, Santa Cruz) antibodies followed by appropriate HRP-conjugated second-stage reagents (for the liver lysates) using ECL detection. For far-western assays, recombinant mouse neuropilin-1 (R&D Systems, 5994-N1) and recombinant mouse VEGF164 (R&D Systems, 493-MV) were spotted onto filters. Both neuropilin-1 and VEGF are known heparin-binding proteins47, 48. The PLVAP–Fc chimaera (in TBS containing 5% BSA) was allowed to bind to the immobilized proteins in the presence or absence of 50 IU heparin (stock 5,000 IU ml−1; Leo Pharma) for 2 h. The bound chimaera was visualized using HRP-conjugated anti-human IgG antibody and ECL. Detection of VEGF interaction with PLVAP in situ in E14.5 fetal livers was performed using a proximity ligation assay (PLA)49. In brief, primary antibodies were rabbit anti-VEGF (46154, Abcam) or rabbit anti-GFP (A11122, Molecular Probes; as a negative control), and they were detected by Duolink in situ PLA probe anti-rabbit PLUS (DUO92002, Sigma), and rat anti-PLVAP antibody (MECA-32) was directly conjugated to MINUS PLA probe using Duolink in situ probemaker MINUS kit (DUO92010, Sigma). After ligation and amplification, the probes were detected using Detection reagent red (DUO92008, Sigma). During the amplification step, Alexa Fluor 488-conjugated donkey anti-rat IgG (A11035, Invitrogen) was added to detect MECA-32. The samples were stained with DAPI and mounted in Mowiol. Images for PLA were acquired using a 3i Spinning Disk confocal microscope (Intelligent Imaging Innovations) with a plan-apochromat 63×/1.4 oil objective and SlideBook 6 software (Intelligent Imaging Innovations). Background subtractions and linear brightness adjustments were performed using ImageJ. Adjustments were applied equally to images captured from control and anti-VEGF antibody PLVAP PLA stains. For co-immunoprecipitation assays, freshly isolated E14.5 livers of wild-type mice were briefly lysed in a buffer containing 1% NP-40, 150 mM NaCl, 20 mM HEPES (pH 7.5), 2 mM MgCl , 2 mM CaCl , PhosSTOP and Protease inhibitor cocktail (both Roche). After clarification by centrifugation, the supernatants were incubated with a rabbit anti-VEGFA antibody (or with a negative control rabbit antibody) for 5 h at 4 °C. Protein G beads (blocked with 1% BSA) were then added for 1 h at 4 °C, and thereafter the beads were washed 3 times with the lysis buffer. The bound proteins were eluted in non-reducing Laemmli’s sample buffer, separated in SDS–PAGE and immunoblotted for VEGF and PLVAP using IRDye-conjugated second-stage reagents and Odyssey imager. Sample size was empirically determined based on pilot analyses and previous literature. Adult wild-type and Plvap−/− littermates were allocated to experimental groups without specific randomization methods because comparisons involved mice of distinct genotypes. The investigators were blinded to the genotype of the embryos during the experimental procedures. Numerical data are given as mean ± s.e.m. Comparisons between genotypes were performed using Mann–Whitney U-test. SAS 9.4 statistical software and GraphPad Prism software v6 were used for statistical analysis. P < 0.05 was considered to be statistically significant. Each data point (values provided in Source Data) is obtained from a different embryo or mouse, and thus all numeric data and statistical analyses are derived from biological replicates.
News Article | February 3, 2016
All cells were grown in media supplemented with 10% fetal calf serum (FCS) (Sigma), 100 IU ml−1 penicillin/streptomycin (Sigma) and 2 mM l-glutamine (Sigma), and grown in a humidified incubator at 37 °C with 5% CO . HAP1 (ref. 8) cells and K562 cells (American Type Culture Collection (ATCC)) were cultured in complete IMDM media. HT29, U2OS (both obtained from ATCC), Caco-2, A549 (both gifts from L. Popov), HEK-293T (from Thermo Scientific), H1-HeLa (from ATCC), HuH7 (a gift from P. Sarnow), mouse embryonic fibroblasts (a gift from K. Storek) and NIH3T3 (a gift from W. Kaiser) cells were all cultured in complete DMEM media. Raji cells (expressing DC-SIGN) (a gift from E. Harris) were cultured in complete RPMI media. The cell lines have not been authenticated or tested for mycoplasma contamination. All isogenic knockout clones were grown in the same media as parent cell lines. HAP1 cells were used for haploid genetic screens (see later). Purified, titred stocks of AAV serotypes 1, 2, 3B, 5, 6, 8 and 9 were purchased from University of North Carolina Chapel Hill Gene Therapy Center Vector Core. These were all self-complementary AAV vectors encoding a reporter fluorescent gene (either GFP or RFP). Purified titred stocks of AAV9–luciferase were also purchased from this core facility to perform mouse experiments. Adenovirus type 5 vector carrying mCherry (Ad5–RFP) was constructed by cloning mCherry cDNA in the pAd/CMV/V5-DEST gateway vector (Invitrogen) according to the manufacturer’s protocol. The following antibodies were used in this study: mouse polyclonal anti-KIAA0319L (ab105385) and rabbit polyclonal anti-giantin (ab24586) (Abcam); rabbit polyclonal anti-TGN46 antibody (NBP1-49643) (Novus Biologicals); mouse monoclonal anti-GAPDH (GT239) (Genetex); rabbit polyclonal anti-FGFR1 (D8E4) and rabbit IgG2a isotype control (Cell Signaling Technology); mouse monoclonal phycoerythrin-conjugated anti-MET antibody (95106) and phycoerythrin-conjugated mouse IgG1 isotype control (R&D Systems, Inc). A high-affinity F-actin, fluorescently labelled probe (Alexa Fluor-660 phalloidin) was used to visualize the cell interior and periphery (Life Technologies). Cells were seeded at 10,000 cells per well (96-well plate) overnight. They were then infected with AAV at a MOI of 20,000 vg per cell (unless otherwise specified) in complete DMEM. Virus infectivity was determined 24 h after infection by measuring transgene expression (RFP, GFP or luciferase) using flow cytometry or bioluminescence. In the case of wild-type AAV2 infection, HeLa wild-type or AAVRKO cells were seeded overnight, then infected with wild-type AAV2 (MOI: 1,000 vg per cell) in the presence of wild-type Ad5 (helper virus). RNA was collected using the Ambion Cell-to-C kit (Thermo Scientific) 24 h after infection, and the generated cDNA was used to perform quantitative reverse-transcriptase PCR (RT–qPCR). mRNA levels of the AAV2-encoded rep68 gene were measured (as a means to detect viral replication) and normalized to 18S ribosomal RNA. Primers against rep68 cDNA included: 5′-CCAATTACTTGCTCCCCAAA-3′ and 5′-CGTTTACGCTCCGTGAGATT-3′. Primers against 18S rRNA included: 5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′. Ad5–RFP was used to infect cells to obtain 50–60% transduction (Fig. 4a), and flow cytometry was used to measure RFP expression. All infections were performed in triplicate, and all data presented are representative of at least two independent experiments. The haploid genetic screen was performed similarly to the protocol described in ref. 8 with minor changes. Briefly, gene-trap virus was used to create a mutagenized HAP1 library. Of this mutagenized library, 100 million cells were infected with AAV2–RFP at a MOI 20,000 of vg per cell. After 48 h, infected cells were sorted by FACS, where RFP-negative cells (approximately 4% of the population) were sorted and grown over a period of 4 days. The resulting sorted cells were then infected again with AAV2 as before, and re-sorted to enrich the RFP-negative (AAV-resistant) population. Thirty-million cells of the resistant population were used for genomic DNA isolation. We performed sequence analysis of gene-trap insertion sites, and the significance of enrichment for each gene in the screen was calculated by comparing how often that gene was mutated and how often the gene carried an insertion in the control data set (owing to random integration). For each gene, a P value was calculated using the one-sided Fisher exact test in R. The P values were corrected for multiple testing according to the Benjamini and Hochberg method (using the R statistical package), to control for false discovery rate26. In the case of KIAA0319L, the P value was lower than the software could report. The numerical value was thus set to 1 × 10−307 (smallest non-zero normalized floating-point number R could report). CRISPR-Cas9 gene editing technology was used to generate isogenic knockout alleles by targeting exonic sequences shared among all protein-coding transcripts of the respective genes as described in ref. 27. The targeted sequences are depicted in Extended Data Table 1, along with the respective mutations. CRISPR sequence targeting oligonucleotides were designed using the Zhang lab CRISPR design tool (http://crispr.mit.edu). Oligonucleotides corresponding to the guide RNA (gRNA) sequences in Extended Data Table 1 were synthesized (Integrated DNA Technologies). gRNA oligonucleotides were directly cloned into Cas9-expressing plasmids pX330 or pX458 (generated by the Zhang lab; obtained from http://www.addgene.org; plasmid 42230 or 48138). Respective cells were transiently transfected with gRNA-encoding plasmids (and GFP-expressing pcDNA vector with gRNA-pX330 plasmids) using Fugene (Promega). After 48 h, GFP-expressing cells were subcloned using the BD InFlux Cell Sorter at the Stanford Shared FACS facility. They were then expanded over 2 weeks and screened genotypically for the mutated allele by extracting genomic DNA from subclones (using the quick DNA universal 96-kit; Zymo Research), amplifying a 500–700 base-pair (bp) region that encompassed the gRNA-targeted site, and sequencing (ElimBio) the resulting PCR product to identify subclones with knockout mutations. The B3GALT6 isogenic knockout clone was generated using TALENs directed against the nucleotide sequence 5′-TGGCCATGCTGGCCTGGCTG-3′, and the reverse complement sequence of 5′-GAGTTCGTGCTCAAGGCGGA-3′ in the only exon of B3GALT6 (transcript ENST00000379198) as described previously28. One day after transfection, cells were selected with blasticidin S (30 μg ml−1, InvivoGen) for 24 h, then stained using anti-heparan sulfate antibody. Cells displaying low staining intensity were subcloned by FACS. To generate the AAVR full-length construct and ΔC-tail, Gibson assembly reaction kit (New England Biolabs) was used to insert the gene of interest into a lentiviral-based vector, pLenti-CMV-Puro-DEST (w118-1) (plasmid 17452), digested with EcoRV to remove the DEST cassette (a gift from E. Campeau)29. AAVR and derived AAVR genes were amplified from a KIAA0319L cDNA clone (clone ID 3843301) (GE Dharmacon), but a single nucleotide polymorphism at position 447 was changed from a ‘T’ to a ‘G’, allowing the sequence to align to the annotated human genome. The following primers were used to generate PCR products from the human KIAA0319L cDNA to be cloned directly into pLent-CMV-Puro-DEST. AAVR full-length: 5′-ATGTGTGGTGGAATTCTGCAGATACCATGGAGAAGAGGCTGGG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTGTAATCCAGGATCTCCTCCCGC-3′; ΔC-tail: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTGTAATCTCCTTTTTGCCTCTTACAAC-3′. Note that reverse primer was designed to incorporate a C-terminal 1× Flag-tag sequence. To generate the AAVR deletion constructs, two or three PCR products were generated using the AAVR construct (with Flag-tag) as a template. They were then assembled into the pLenti-CMV-Puro-DEST vector using the Gibson assembly reaction. Primers used to amplify the N-terminal fragments for the following constructs were: ΔMANEC: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTCACTGGCATCTGTTGAC-3′; ΔPKD1–2: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CAGTTCCTTTATAACTGGGTATGG-3′; ΔPKD2–3: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTACGGGGCTCTGGC-3′; ΔPKD3–4: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-GTAATCCACAGCTTTG TTCAC-3′; ΔPKD4–5: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′; miniAAVR: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTCACTGGCATCTGTTGAC-3′; middle fragment of miniAAVR: 5′-GTCAACAGATGCCAGTGAGGTATCTGCTGGAGAGAGTGTC-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′. Primers used to amplify the C-terminal fragments for the following constructs were: ΔMANEC: 5′-GTCAACAGATGCCAGTGAGACACACTCCTCCAATTCCAT-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD1–2: 5′-CCATACCCAGTTATAAAGGAACTGCCCCCTGTGGCCAACG-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD2–3: 5′-GCCAGAGCCCCGTAAGCCTCCTCAGGCAGATGC-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD3–4: 5′-GTGAACAAAGCTGTGGATTACCCACCTATAGCCAAGATAACTG-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD4–5: 5′-ATTGTGCAACCTGAAAACAATAAGAACCTGGTGGAGATCATCTTGGATATC-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; miniAAVR: 5′-ATTGTGCAACCTGAAAACAATAAGTGTGAGTGGAGCGTGTTATATG-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′. AAVR PKD domains 1–5 (residues 311–787) were expressed in E. coli using the pMAL expression system (New England Biolabs). A bacmid, created from a pFastBac dual vector containing the cDNA for the KIAA0319L ectodomain fused to a C-terminal influenza haemagglutinin (HA)-tag was a gift from M. van Oers, and obtained with the assistance of M. Waye30. cDNA coding for PKD domains 1–5 was cloned out of the pFastBacDual expression vector and inserted into the pMAL-c5X vector, using 5′-GTATCTGCTGGAGAGAGTGTCCAGATAACC-3′ and 5′-CAGGTTGTTTTTCCTGCAGGTCACCTGGGATCAGGTTTCAC-3′, then expressed in NEBexpress cells (New England Biolabs). This resulted in a fusion protein comprised of a mannose-binding protein (MBP) tag and AAVR PKD domains 1–5 (referred to as: soluble AAVR). MBP was specifically used as an affinity tag for ease of purification. To create AAVR fusion constructs, Ci-MPR-tail, LDLR-tail and PVR-tail, the Gibson assembly reaction was used to fuse amplified miniAAVR without its C-terminal to the C-terminal of the respective proteins, and insert it into the pLenti-CMV-Puro-DEST vector. Primers used for amplification and insertion included: miniAAVR without C-terminal and transmembrane domain for Ci-MPR-tail: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′; MPR C-terminal and transmembrane: 5′-ATTGTGCAACCTGAAAACAATAAGGCTGTGGGAGCTGTGC-3′ and 5′-CGGCCGCCACTGTGC-3′; miniAAVR without C-terminal and transmembrane domain for LDLR-tail or PVR-tail: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′; LDLR or PVR C-terminal and transmembrane: 5′-ATTGTGCAACCTGAAAACAATAAG-3′ and 5′-TAAATCCAGCACAGTGGCGGCCG-3′. Lentiviral transduction was used to create stable cell lines expressing a selected gene of interest under a CMV promoter. Using Gibson assembly reaction, the respective genes of interest (see ‘construction of plasmids’ section) were inserted into the pLenti-CMV-Puro-DEST vector, and used as described previously29. Lentivirus was produced using HEK293 cells and used to transduce the respective cell lines overnight. Cells stably expressing the gene of interest were selected by treatment with 1–3 μg ml−1 puromycin over 2 days (InvivoGen). A lentivirus carrying the mCherry (RFP) gene was used as a control for AAVR complementation in AAVRKO cells. All flow cytometry was performed at the Stanford Shared FACS facility. To perform the haploid genetic screen, FACS was carried out on a FACS Aria flow cytometer (BD). To measure virus transgene expression (RFP/GFP) in all other experiments, cells were trypsinized 24 h after infection and a LSRII-UV flow cytometer (BD) was used to detect fluorescent cells. For cell surface staining, cells were trypsinized and washed using FACS buffer (PBS supplemented with 2% FCS, 1 mM EDTA and 0.1% sodium azide). They were subsequently incubated for 40 min at 4 °C with the respective primary antibodies at a 1:50 dilution (see ‘Antibodies’ section), washed, and incubated for a further 40 min at 4 °C with Alexa488- or Alexa594-conjugated secondary antibodies (1:500 dilution; if the primary was not conjugated) (Life Technologies). This was followed by a final wash and resuspension of cells in FACS buffer before reading fluorescence. All data presented are representative of at least two independent experiments. Data were analysed and assembled using FlowJo software (TreeStar Inc). Cell pellets of 2 × 106 cells were lysed with Laemmli SDS sample buffer containing 5% β-mercaptoethanol and boiled for 10 min at 96 °C. Lysates were separated by SDS–PAGE using the Mini-Protean system (Bio-Rad) on 4–15% polyacrylamide gradient gels (Bio-Rad). Proteins were transferred onto polyvinylidene fluoride membranes (Bio-Rad) using the Bio-Rad Transblot protein transfer system in a semi-wet preparation. Membranes were blocked by incubating with PBS containing 5% non-fat milk for 1 h at room temperature. Membranes were subsequently incubated overnight at 4 °C with primary antibodies at a dilution of 1:1000 (anti-KIAA0319L antibody) or 1:2,000 (anti-GAPDH antibody) in blocking buffer. Membranes were washed 3 times for 5 min using wash buffer (PBS with 0.1% Tween-20), and further incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse and anti-rabbit 1:5,000 in blocking buffer) (GeneTex) for 1 h at room temperature. After another set of three washes, antibody-bound proteins were visualized on film using the West Pico and Extended Duration chemiluminescence peroxide solutions (Thermo Scientific). Cells were seeded overnight at 40,000 cells per well onto LabTekII glass chamber slides (Thermo Scientific). They were washed once with PBS, and either treated or fixed immediately with 4% paraformaldehyde for 15 min. They were washed three times with PBS before being incubated for 1 h at room temperature with primary antibodies against the respective proteins at a dilution of 1:100 (anti-KIAA0319L and anti-TGN46) or 1:200 (anti-giantin) in immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin and 1% Triton X-100). Cells were then washed three times in PBS, and incubated for a further hour in DAPI stain (1:500) and fluorescently tagged secondary antibodies (Alexa488 anti-mouse and Alexa594 anti-rabbit; Life Technologies) at a dilution of 1:300. Cells were washed a final three times in PBS, and 5 μl of Vectashield (Vector Laboratories Inc) was applied to each slide chamber before a glass cover slip (VWR International) was placed over slide to mount samples. Cells were visualized directly with a Zeiss LSM 700 confocal microscope. Purification of the soluble AAVR was achieved through amylose-based MBP affinity chromatography (GE Healthcare). ELISA plates (Corning Costar) were coated overnight at 4 °C with 50 μl AAV2 virus-like particles at 2.5 μg ml−1 in 100 mM NaHCO (pH 9.6). Plates were then washed with TBST buffer (0.05% Tween-20 in TBS) and blocked with 3% BSA in TBST for 1 h at room temperature. Subsequent washing was followed by incubation with soluble AAVR or MBP control at the indicated concentrations for 2 h at room temperature. Anti-MBP–HRP (1:500, 1 h incubation at room temperature) was used to detect soluble AAVR and MBP controls, requiring no secondary antibody. Samples were developed with 1-Step Ultra TMB-ELISA substrate as per the manufacturer’s instructions (Thermo Scientific) and optical density assayed by microplate reader (Molecular Devices SpectraMax M2e) at 450 nm. Curve fitting was performed in SigmaPlot v12.5 (Systat Software, Inc). All data presented are representative of at least three independent experiments. Surface plasmon resonance analysis was carried out using a BIAcore X instrument (GE Healthcare) and a flow rate of 10 μl min−1 at 20 °C in HBS-P buffer (10 mM HEPES (pH 7.5), 150 nM NaCl and 0.005% surfactant P20). His-tagged soluble AAVR (His-tagged MBP fusion with AAVR PKD domains 1–5) at various concentrations was mixed with His-tagged MBP to a total concentration of 0.2 μM in 10 mM sodium acetate buffer (pH 4.0) and immobilized on a CM5 sensor chip by amide coupling. MBP at 0.2 μM was sufficient to block nonspecific binding to the dextran. For the analysis of binding affinity, all curves were measured in triplicate and were fitted with a Langmuir 1:1 binding model (BIAevaluation software, GE Healthcare). Wild-type HeLa cells were seeded in 96-well plates at 10,000 cells per well overnight. Anti-AAVR antibody (ab105385) or IgG isotype control (both from Abcam) were incubated with cells (at concentrations ranging from 0.5 to 50 μg ml−1 in DMEM media) for 1 h at 4 °C. Cells were then infected with AAV2–luciferase at a MOI of 1,000 vg per cell, and left for 24 h at 37 °C. A luciferase assay kit (E1500, Promega) was used to detect bioluminescence, with measurements being taken on the Promega GloMax luminometer. Notably, the storage buffers of both antibodies did not contain preservatives such as azide that could interfere with the assay. All data presented are representative of two independent experiments. HeLa cells were seeded in 96-well plates at 10,000 cells per well overnight. Purified soluble AAVR or MBP control was then introduced to the medium at the specified concentrations. Cells were transduced with AAV2–GFP at a MOI of 7,500 vg per cell and incubated for 24 h at 37 °C. This was followed by trypsinization and measuring transgene expression by flow cytometry. For immunofluorescence imaging, the concentration of soluble AAVR and MBP controls was 0.1 μM, and transduction was performed using 7,000 vg per cell. At 24 h post-transduction, cells were incubated with 1 μg ml−1 Hoechst stain (Thermo Scientific) in PBS for 10 min at 37 °C, before washing with PBS and subsequent fluorescent imaging (Nikon Eclipse Ti-E). All data presented are representative of two independent experiments. These experiments were performed similarly to Ci-MPR tracking assays, as described in ref. 31. AAVRKO cells with or without overexpression of AAVR or ΔC-tail were incubated at 4 °C with anti-AAVR antibodies (approximately 25 μg ml−1) for 1 h. Cells were then washed three times with PBS and transferred to 37 °C for specific time points (2, 10, 30 and 60 min), at which time they were fixed with 4% paraformaldehyde for 15 min. Following fixation, immunofluorescence staining (as described earlier) was performed to visualize AAVR endocytosis. All data presented are representative of two independent experiments. All the experiments involving animals were conducted in strict accordance with the Institutional Animal Care and Use Committee of Stanford University. Mice were housed in a Stanford University vivarium, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Mice were housed in irradiated disposable caging (Innovive Inc) with bi-weekly cage changes. Mice were provided with irradiated food and ultraviolet-irradiated acidified water. Health surveillance was performed via trimester testing of dirty bedding CD1 sentinels (Charles River Laboratories). Sentinels were consistently negative for mouse parvovirus, minute virus of mice, mouse hepatitis virus, rotavirus, mouse encephalomyelitis virus, Sendai virus, mouse adenovirus 1 and 2, ectromelia virus, lymphocytic choriomeningitis virus, pneumonia virus of mice, reovirus 3, Mycoplasma pulmonis, and endo- and ectoparasites. No statistical methods were used to predetermine sample size. In our animal study protocol, we state that the number of animals in each experimental group varies, and is based on similar previous study32. Randomization was not used to allocate animals to experimental groups and the investigators were not blinded to allocation during experiments and outcome assessment. TALEN technology was used to create AAVR isogenic knockout FVB mice (purchased from Cyagen Biosciences). TALEN-targeted sequences were 5′-TGGGAGTCAAGCCAAGTC-3′ and 5′-GCCAGGATATTGTTGGCAGA-3′. Two founder males were mated to FVB/NCrl (Charles River Laboratories) females. After three rounds of breeding, wild-type (Aavr+/+), heterozygous (Aavr+/−) and homozygous AAVRKO (Aavr−/−) mice were generated, determined by genotyping. All genotypes (wild-type, heterozygous and knockout) were obtained in the expected Mendelian ratios after breeding. At 5 weeks of age, 10 female and 9 male animals were used to examine the effect of Aavr KO on AAV infection in vivo. Animals from each group (Aavr+/+, n = 7 (2 litter mates and 5 purchased FVB mice); Aavr+/−, n = 4; Aavr−/−, n = 4 and uninfected mice, n = 4) were injected intraperitoneally with 1 × 1011 viral genomes of AAV9–luciferase in 200 μl of PBS. All of the mice recovered from the injection quickly without loss of mobility or interruption of grooming activity. Aavr+/+ and Aavr−/− mice were found to be significantly different in two independent experiments. The mice were anaesthetized with 2% isofluorane and oxygen. The d-luciferin substrate (Biotium) was injected intraperitoneally (3.3 μg per mouse). After 10 min, the mice were then placed in a light-tight chamber, and images were generated using a cryogenically cooled charge-coupling device camera IVIS 100 (Xenogen), recording bioluminescence at 1, 10, 60 and 100 s. The visual output represents the average radiance as the number of photons emitted per second per cm2 as a false colour image where the maximum is red and the minimum is dark blue. All animals were imaged on a schedule of 3, 7, 10 and 14 days after AAV vector injection. At each time-point a ‘region of interest’ was designated surrounding each animal in order to quantify the radiance (photons s−1 cm−2 sr−1) being released by luciferase activity. This region was kept the same for each mouse and at each time point. The mean and standard deviation of radiance measurements were determined for each mouse group at each time point. The unpaired parametric two-sided Student’s t-test was used for statistical calculations involving two group comparisons in all tissue-culture-based experiments (*P < 0.05, **P < 0.01, ***P < 0.001), with a Welch post-correction accounting for different standard deviations. An unpaired two-sided Mann–Whitney t-test was used for statistical calculations involving two group comparisons in in vivo experiments. GraphPad Prism was used for statistical calculations.
News Article | April 20, 2016
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.