One of these, TCR-5, exhibited tetramer binding in both CD4 and CD8 cells and was selected for further studies. Antigen-specific and HLA-A*0201-restricted interferon-γ release, cell lysis and lymphocyte proliferation was observed following culture of TCR engineered human PBL with relevant tumor cell lines. Codon optimization was found to increase TCR-5 expression in transduced T cells, and this construct has been selected for development of clinical grade viral vector producing cells. The tumor-specific pattern of expression of SSX2, along with the potent and selective activity of TCR-5, makes this TCR an attractive candidate for potential TCR gene therapy to treat multiple cancer histologies.The clinical success of adoptive immunotherapy of cancer relies on the selection of target antigens that are highly expressed in tumor cells but absent in essential normal tissues. A group of genes that encode the cancer/testis or cancer germline antigens have been proposed as ideal targets for immunotherapy due to their high expression in multiple cancer types and their restricted expression in immunoprivileged normal tissues. In the present work we report the isolation and characterization of human T cell receptors (TCRs) with specificity for synovial sarcoma X breakpoint 2 (SSX2), a cancer/testis antigen expressed in melanoma, prostate cancer, lymphoma, multiple myeloma and pancreatic cancer, among other tumors. We isolated seven HLA-A2 restricted T cell receptors from natural T cell clones derived from tumor-infiltrated lymph nodes of two SSX2-seropositive melanoma patients, and selected four TCRs for cloning into retroviral vectors. Peripheral blood lymphocytes (PBL) transduced with three of four SSX2 TCRs showed SSX2 The synovial sarcoma X breakpoint (SSX) genes are located on the X chromosome and encode a family of ten highly homologous nuclear proteins, SSX1–10. SSX2 was originally identified as part of a genomic translocation present in synovial sarcoma 41-49, isolated from previously described melanoma patients displaying active immune responses to SSX2 We generated retroviral vectors encoding the TCR alpha- and beta-chains targeting the HLA-A*0201-restricted epitope SSX22.HLA-A*0201+/SSX2+ melanoma cell line 624, and non-HLA-A*0201 cell lines 888 and 938 were established from surgically resected metastatic melanoma tumors and maintained at the Surgery Branch, National Cancer Institute, National Institutes of Health . The HLA-A*0201+/SSX2+ glioma cell line U251 was obtained from the Division of Cancer Treatment and Diagnosis Tumor Repository, National Cancer Institute, National Institutes of Health . Melanoma lines SKmel23 and SKmel37, and breast cancer cell line MCF7 were purchased from ATCC . Cos7-A*0201 and 293-A*0201 cells were retrovirally engineered to express HLA-A*0201 as described previously Peripheral blood lymphocytes used in this study were obtained from melanoma patients treated in the Surgery Branch, National Cancer Institute, National Institutes of Health, on NCI Institutional Review Board-approved protocols. Patients provided written consent for procurement of tissues and use of these for research purposes, as detailed in protocol 03-C-0277. Human lymphocytes were maintained in AIM-V medium  supplemented with 5% human AB serum , 50 U/mL penicillin, 50 μg/mL streptomycin (Invitrogen), and 300 IU/mL IL-2 and maintained at 37°C with 5% CO2. SSX2 specific T cell clones were newly generated from tumor infiltrated lymph node-derived T cells of patients Lau 567 and Lau 672, similarly as described previously 41-49 peptide (KASEKIFYV) corresponding to amino acids 41 to 49 of SSX2, and homologous peptides derived from SSX1 (KYSEKISYV), SSX3 (KVSEKIVYV), SSX4 (KSSEKIVYV), SSX5 (KASEKIIYV), SSX6 (KFSEKISCV), SSX7 (KSLEKISYV), SSX8 (KYSEKISYV), SSX9 (KSSEKIIYV), SSX10 (KASEKILYV), IGSF22 (KESAKIFYD), ARHGAP1 (KFGQKIFYV), GPR82 (SCYEKIFYG), PHF8 (LKGEKIFYL), LIPM (TGQEKIYYV), SYT14 (IVGEKIFYL), TCOF1 (KASEKILQV), RBL2 (SPREKIFYY) and FRAS1 (SPREKIYYV) were synthesized by GenScript . Peptides were dissolved in DMSO and diluted in RPMI 1640 medium for loading of T2 cells. Binding affinity to HLA-A2*0201 was predicted for each peptide using NetMHC-3.0 41-49 with a potential for cross-reactivity was performed by blast search .The SSX2MSGV1-based retroviral vectors were constructed by overlapping PCR with the alpha- and beta-TCR chains arranged in the following order: TCR alpha-chain, linker peptide furin-SGSGP2A, TCR beta-chain, as previously described Retroviral supernatants were generated by transfecting each pMSGV1-SSX2-TCR plasmid along with a vector encoding RD114 envelope into 293-GP cells using the Lipofectamine 2000 reagent (Invitrogen) in Opti-MEM medium (Invitrogen) 41-49 (KASEKIFYV) were produced by the National Institutes of Health Tetramer Core Facility at Emory University  using phycoerythrin (PE) as the fluorochrome. For evaluation of TCR transduction efficiency in T cell subsets, transduced T cells were stained with a FITC-labeled anti-human CD8  and with PE-labeled HLA-A*0201 tetramers. Cells were analyzed using a FACScan flow cytometer with CellQuest software (BD Biosciences) or FlowJo software .HLA-A*0201–restricted SSX2–derived peptide SSX25 of each) in 200 uL of AIM-V medium in duplicate wells of a 96-well microplate. Culture supernatants were harvested 18–24 h after the initiation of coculture and assayed for interferon-γ (IFNg) by ELISA (Thermo Scientific).TCR-transduced lymphocytes were tested for antigen-specific reactivity in cytokine release assays using peptide-loaded T2 cells or tumor cells. To that end, effector cells and target cells were cocultured at a 1∶1 ratio (1×1051Cr] release assay as described previously 51Cr-labeled target cells (E:T ratio) in AIM-V medium in 96-well U plates at 37°C for 4 h. Lysis was measured by [51Cr] release in the medium according to the formula: percent lysis  =  (sample release - minimum release)/(maximum release - minimum release) x 100%. Results expressed as average of duplicate samples.The ability of the transduced PBLs to lyse HLA-A*0201+ SSX2+ tumor cells was assessed using a standard thymidine as average counts of triplicate wells. Co op: codon optimized; MCR: codon-optimized plus mouse constant region. B) Flow cytometry analysis of CD137 (4-1BB) expression in TCR-5-transduced T cells upon coculture with HLA-A*0201+ SSX2+ or HLA-A*0201+ SSX2- cells . Staining with anti-CD8 and anti CD137 antibodies was performed after an overnight coculture. C) Flow cytometry analysis of IFNg and IL-2 expression in TCR-5-transduced T cells upon coculture with HLA-A*0201- SSX2+ or HLA-A*0201+ SSX2+ cell lines . Gated on CD3+ cells.(TIF)Click here for additional data file.Methods S1(DOCX)Click here for additional data file.


Caenorhabditis elegans PKG ortholog, modulates foraging behavior and egg-laying and seeks the downstream effectors of EGL-4 activity. Using a combination of unbiased forward genetic screen and proteomic analysis, we have identified a conserved SAEG-1/SAEG-2/HDA-2 histone deacetylase complex that is specifically recruited by activated nuclear EGL-4. Gene expression profiling by microarrays revealed >40 genes that are sensitive to EGL-4 activity in a SAEG-1–dependent manner. We present evidence that EGL-4 controls egg laying via one of these genes, Y45F10C.2, which encodes a novel protein that is expressed exclusively in the uterine epithelium. Our results indicate that, in addition to cytoplasmic functions, active EGL-4/PKG acts in the nucleus via a conserved Class I histone deacetylase complex to regulate gene expression pertinent to behavioral and physiological responses to cGMP. We also identify transcriptional targets of EGL-4 that carry out discrete components of the physiological response.Elevation of the second messenger cGMP by nitric oxide (NO) activates the cGMP-dependent protein kinase PKG, which is key in regulating cardiovascular, intestinal, and neuronal functions in mammals. The NO-cGMP-PKG signaling pathway is also a major therapeutic target for cardiovascular and male reproductive diseases. Despite widespread effects of PKG activation, few molecular targets of PKG are known. We study how EGL-4, the  C. elegans. Using a combination of unbiased forward genetic screen and proteomic analysis, we show that constitutively active EGL-4 alters gene expression in multiple tissues, which is achieved through activity-dependent recruitment of a conserved Class I histone deacetylase complex in the nucleus. Furthermore, we identify a novel EGL-4–responsive gene that encodes a putative secreted protein that modulates the egg laying rate of C. elegans. Taken together, our results uncover novel PKG targets in the nucleus that respond to sustained elevation of cGMP. Development of chemicals that modulate the activity of these PKG targets may differentiate or alleviate undesirable side-effects of existing drugs that manipulate cGMP level.Nitrates and phosphodiesterase inhibitors raise the intracellular level of cGMP, and they have been widely used to treat hypertension and erectile dysfunction. Although it is known that cGMP activates the cGMP-dependent protein kinase PKG, which in turn causes smooth muscle relaxation and other physiological responses, very few molecular targets of PKG have been identified. In addition, the long-term effects of sustained elevation of cGMP and PKG activation are not known. We study a family member of PKG called EGL-4 in the nematode  C. elegans PKG ortholog, EGL-4, in order to discover additional evolutionarily conserved molecular effectors of cGMP-PKG singaling.The cellular level of cGMP is controlled by a balance of guanylyl cyclase and phosphodiesterase activities C. elegans. Animals navigate towards food source and away from unfavorable growth conditions, such as sub-optimal temperature, high population density and pathogenic organisms C. elegans, insulin, TGF-β and cGMP signaling pathways have been implicated in organismal homeostasis in response to changes in environmental conditions C. elegans.Environmental conditions dictate larval developmental decisions and a number of adult behaviors such as foraging and egg laying in the free living nematode C. elegans cGMP dependent protein kinase EGL-4 were originally identified from a genetic screen for egg laying defective mutants Drosophila as the expression level of the PKG ortholog foraging dictates sitter versus rover phenotype in larvae Mutant alleles of the C. elegans EGL-4 is most homologous to mammalian PKG-Iβ, which has an auto-inhibitory domain at its N-terminus, followed by two cGMP binding domains and a kinase domain The C. elegans mutant that expresses a constitutively active EGL-4/PKG. Our analyses revealed genomic effects of EGL-4 activity via a conserved histone deacetylase complex. We also present evidence that EGL-4 activity can modulate the expression of a novel extracellular signaling protein, which illustrates how EGL-4 may exert cell non-autonomous effects.To address how EGL-4 and its downstream effectors may coordinate physiological responses upon changes in environmental conditions, we characterized a mg410, a dominant egl-4 gain-of-function allele from a genetic screen for mutants with elevated Nile Red staining in the absence of a functional peroxisomal thiolase, kat-1egl-4(mg410) mutant animals display pleiotropic phenotypes such as excessive dwelling and reduced body length  suppressor alleles. Among them, hj32, a null allele that encodes a pre-mature stop codon for a complete loss of EGL-4 protein  and egl-4(mg410hj40) mutant animals are phenotypically similar to wild-type animals  mutant are due to developmental defects of specific neuronal circuits. Alternatively, EGL-4 may regulate foraging behavior and egg laying frequency by affecting the activity of pre-established circuits. To distinguish between these possibilities, we transiently expressed a constitutively active (K162N) or kinase dead (K499A) form of EGL-4 that carries a FLAG-epitope tag under the control of a heat-shock promoter in wild-type animals. These animals were allowed to develop to young adult stage when all neuronal circuits are established. After heat-shock, the FLAG-tagged EGL-4 reached steady-state level in 2 hours and started waning after 12 hours (hjIs28) or kinase dead EGL-4 (hjIs30), 2 hours after heat-shock when the expression level of ectopic EGL-4 peaked (hjIs28) caused excessive dwelling when compared with wild-type animals and the kinase dead control (hjIs30) 24 hours after heat-shock (egl-4(gf) mutant animals. The same temporal effect was observed on egg laying frequency upon ectopic expression of a constitutively active EGL-4 (egl-4(gf) animals were not due to a defect in neuronal circuit assembly. Given the delayed effects of EGL-4 activation, we hypothesize that EGL-4 may mount a transcriptional program that triggers sustained changes on established neuronal circuits. Nevertheless, we cannot rule out a role for EGL-4 in regulating short-term synaptic activity.Foraging behavior and egg laying rate are controlled by dedicated neuronal circuits that couple sensory inputs with motor outputs. It is plausible that the pleiotropic phenotypes of the 12 hours . Express12 hours . Using t4 peaked . The perat-shock . The excve EGL-4 . We concegl-4(gf) animals for mutant alleles that suppressed their excessive dwelling behavior. These alleles fell into two complementation groups, defining two suppressor of activated EGL-4 (saeg-) genes. Several lines of evidence suggest that saeg-1 and saeg-2 are likely to encode ubiquitous downstream effectors of EGL-4. First, recessive mutations in saeg-1 and saeg-2 genes strongly suppressed excessive dwelling, accelerated egg laying rate and short body length of egl-4(gf) animals ; saeg-1, egl-4(gf); saeg-2 and egl-4(gf); saeg-1; saeg-2 mutant animals  coding sequence immediately 5′ to the saeg-1 stop codon. We found that SAEG-1::GFP is a ubiquitous nuclear protein  animals.We isolated four recessive alleles of  protein . The zinsaeg-2 gene was defined by a single recessive allele, hj9, from our genetic screen. The hj9 allele encodes a guanine to adenine substitution that eliminates the consensus splice donor site of the third intron. A second allele of saeg-2, ok3174, was isolated by the C. elegans Knockout Consortium. Both hj9 and ok3174 alleles are molecular null alleles because no SAEG-2 protein is detectable in animals carrying either alleles, which show comparable phenotypes  animals specifically associated with SAEG-2  or Dnttip1 (SAEG-2 ortholog). Immunoprecipitation was followed by Multi-Dimensional Protein Identification Technology (MuDPIT) analysis C. elegans HDA-2 is the closest ortholog of mammalian HDAC1 and HDAC2 and it co-immunoprecipitated with SAEG-2 when over-expressed in Drosophila S2 cells  animals (egl-4(gf) phenotypes by loss of hda-2 function could be reversed with an hda-2p::hda-2(+) transgene, demonstrating the specificity of such genetic interaction  phenotypes  animals in a SAEG-1/SAEG-2 dependent manner. These genes should also be de-repressed or activated in egl-4(lf) animals. Therefore, we performed gene expression profiling using Affymetrix microarrays on RNA samples extracted from wild-type, egl-4(gf), egl-4(lf) and egl-4(gf); saeg-1(lf) mutant animals at the late larval L4 and young adult stage. By focusing on genes that showed >1.5-fold change in expression (p<0.05) when compared with wild-type samples, we identified 60 genes that were repressed in egl-4(gf) and activated in egl-4(lf) animals (egl-4(gf); saeg-1(lf) animals. In addition, 5 out of these 60 genes were de-repressed and activated (>1.5-fold) in the same animals. These results demonstrate that EGL-4 modulates gene expression almost entirely through SAEG-1, and by inference the SAEG-1/SAEG-2 complex. Although we have identified a group of genes whose expression was highly responsive to EGL-4 activity, our results did not imply direct recruitment of the SAEG-1/SAEG-2 complex to their promoters.Our results point to a molecular pathway in which activated EGL-4/PKG kinase engages a conserved histone deacetylase complex that modulates expression of key regulators of foraging behavior and egg laying rate. We hypothesized that such EGL-4 activity responsive genes are most likely repressed in  animals . The expegl-4(gf) versus egl-4(lf) animals (egl-4(gf); saeg-1(lf) animals. We verified the gene expression changes in Y45F10C.2 by quantitative real-time PCR  animals where Y45F10C.2 expression was high (egl-4(gf) animals, consistent with the observation that the endogenous Y45F10C.2 gene was highly repressed in these animals . The molecular target of Y45F10C.2 is unknown. It is plausible that Y45F10C.2 can bind to novel cell surface receptors that inhibit egg laying or modulate the function of serotonin, acetylcholine or neuropeptide receptors at the neuromuscular circuit for egg laying.How does EGL-4 regulate egg-laying has been an open question since the original isolation of in vivo role for the mammalian orthologs of SAEG-1 and SAEG-2. In cell culture systems, TRERF1 has been reported to activate CYP11A1, a gene required for steroidogenesis C. elegansOur results support a surprising While the intracellular cGMP level is tightly regulated by the opposing action of guanylyl cyclase (GC) and phosphodiesterase (PDE) physiologically C. elegans, we expect to identify additional EGL-4 responsive genes that regulate foraging behavior and body length. It is plausible that the SAEG-1/SAEG-2 histone deacetylase complex may engage tissue specific transcription factors, which may be identified through isolation of tissue specific genetic suppressors of the egl-4(gf) mutant. Given the deep conservation of EGL-4, SAEG-1 and SAEG-2, our model should be readily applicable in mammals and expand the mode of action of cGMP signaling and its downstream kinase PKG.In summary, our results provide a molecular framework on how nuclear EGL-4/PKG activity triggers wide-spread physiological responses. In The wild-type strain was Bristol N2. All animals were raised at 20°C. The following alleles and transgenes were used:hda-3(ok1991)LGI: hda-2(ok1479)LGII: saeg-2(hj9), saeg-2(ok3174)LGIII: egl-4(n479), egl-4(mg410), egl-4(mg410hj32), egl-4(mg410hj40), egl-4(mg410hj33)LGIV: saeg-1(hj11), saeg-1(hj12), saeg-1(hj15), saeg-1(hj16)LGV: hda-4(ok518)LGX: hjSi15[saeg-2p::saeg-2::GFP-3xFLAG]Generated by MosSCI, outcrossed 2 times with N2.hjIs28[hsp-16-41p::3xFLAG::EGL-4(K162N)::SL2::mCherry::C. briggsae unc-119(+)]Generated by micro-particle bombardment, outcrossed 2 times with N2.hjIs30[hsp-16-41p::3xFLAG::EGL-4(K499A)::SL2::mCherry::C. briggsae unc-119(+)]Generated by micro-particle bombardment, outcrossed 2 times with N2.hjIs46[Y45F10C.2p::Y45F10C.2::SL2::GFP-PEST]Generated by UV irradiation, outcrossed 5 times with N2.hjEx6, hjEx7 [hda-2p::hda-2::SL2::GFP] (5 ng/µl)mec-7::rfp (30 ng/µl) and pBluescript (65 ng/µl) injected intohda-2(ok1479); egl-4(mg410)hjEx8, hjEx9 [Y45F10C.2p::Y45F10C.2(C36A)::SL2::GFP] (50 ng/µl)mec-7::rfp (30 ng/µl) and pBluescript (20 ng/µl) injected into N2hjEx10 [saeg-1p::saeg-1::GFP::saeg-1 3′UTR] encompassing the 5′ regulatory region and coding sequence of saeg-1 from fosmid WRM062aH08 at 5 ng/µl; 8.3 kb ApaI/BamHI fragment (coordinates: 10851160–10858568) from fosmid WRM062aH08 encompassing the 3′ coding sequence and 3′ UTR of saeg-1 with GFP coding sequence inserted immediately 5′ to the stop codon at 1.8 ng/µl)mec-7::rfp (10 ng/µl) and myo-2::mCherry (2.5 ng/µl) injected intoegl-4(mg410); saeg-1(hj12)egl-4(mg410) allele was isolated in a previously described genetic screen for mutants that enhanced Nile Red staining of kat-1(mg368) mutant animals C. elegans isolate CB4856 mg410 to LGIV between snp_Y38C1BAegl-4 and identified a single G to T mutation that introduced a K162N (residue numbering as in EGL-4A) substitution in animals carrying the mg410 allele. This mutation is located in a common exon shared by isoforms a, b, c, and e of egl-4.The egl-4(mg410), we mutagenized egl-4(mg410) animals with ethyl methane-sulfonate (EMS) by using standard procedures. We screened 34,000 haploid genomes and retrieved both intragenic and extragenic suppressors of egl-4(mg410), which displayed an increase in body length and roaming behavior. Intragenic suppressors are egl-4 loss-of-function alleles, including hj32, hj33 and hj40. In addition, we isolated extragenic suppressor alleles that fell into two complementation groups. Genetic mapping with the Hawaiian isolate CB4856 placed saeg-2(hj9) on LGIII between snp_F54F2saeg-2 promoter from a single copy transgene (hjSi15) also rescued egl-4(mg410); saeg-2(ok3174) animals. However, over- expression of SAEG-2::GFP from extra-chromosomal transgenes failed to do so. We mapped saeg-1(hj11) to LGV between snp_T21C9hj11, hj12, hj15 and hj16 and we rescued animals carrying hj12 by injecting the fosmid WRM062aH08, which contained only the coding and regulatory sequences of F53H10.2. We used hj12 as the reference allele, which introduced a pre-mature stop codon in place of Arg615 (residue numbering as in SAEG-1A) and an 80% decrease in mRNA level as measured by quantitative real-time PCR .To isolate genetic suppressors of hjIs28 and hjIs30 young adult animals as judged by vulval morphology. Animals were raised at 20°C on NGM agar plates seeded with OP50 and were heat shocked for 30 mins at 33°C, before returning to 20°C for defined periods of time. 50 animals were picked at each time point for detection of EGL-4 protein by Western blotting.Heat-shock experiments were performed with synchronized wild-type, Antibodies against EGL-4 were raised in rabbits using a GST-EGL-4A(32-215) fusion protein. EGL-4A(32-215) encompasses the auto-inhibition domain, which is also present in EGL-4 isoforms B, C and E. Antibodies against SAEG-2 were raised in rabbits using full-length SAEG-2 fused to the Pseudomonas Endotoxin at the N-terminus and a poly-histidine tag at the C-terminus. Monoclonal antibodies against the FLAG epitope  and HA epitope  were used for Western blot. For immunoprecipitation, anti-FLAG  and anti-HA  antibodies conjugated to agarose beads were used.Drosophila S2 cells were transfected with copper inducible expression plasmids based on pMT-V5-HisB (Invitrogen) using Effectene Transfection Reagent (Qiagen). 24 hrs after transfection, 700 µM copper sulfate was added to induce gene expression. Cells were harvested 24 hrs later and whole cell lysates were prepared using MAPK buffer . Human embryonic kidney (HEK293) cells were transfected with expression plasmids based on pcDNA5 (Invitrogen) using Lipofectamine2000 (Invitrogen). Cells were harvested 24 hrs after transfection and whole cell lysates were prepared using MAPK buffer. Immunoprecipitations with agarose beads conjugated with anti-FLAG or anti-HA antibodies were carried out at 4°C for 2.5 hrs.E. coli HB101 were used for preparation of worm lysates and immunoprecipitation as described Mixed-stage worms grown in liquid culture with Drosophila S2 cells. After immunoprecipitation, anti-FLAG agarose beads were washed 4 times with MAPK buffer and once with kinase buffer ). In vitro phosphorylation was allowed to proceed in the presence of 1 µCi 32P ATP in kinase buffer at 30°C for 20 mins. Agarose beads were washed 4 times with kinase buffer and bound proteins were released by boiling in LDS sample buffer (Invitrogen), separated by SDS-PAGE and visualized by autoradiography.Autophosphorylation of EGL-4 was monitored using FLAG-tagged EGL-4 that was expressed in E. coli OP50 on the entire surface. Movies were taken on a Lumar dissecting microscope (Zeiss) equipped with a CCD camera with a field of view of 2.2 cm×1.7 cm. Animals that moved out of the field were defined as new objects by the Worm Tracker program. We assigned an animal as dwelling if it exhibited angular velocity ≥110°/s or ≤ –110°/s, for a duration ≥30 s. We assigned an animal as roaming if it was not engaged in dwelling as described above.For automated worm tracking using the Worm Tracker program E. coli OP50 (∼2 cm diameter) at 20°C. An image of the bacterial lawn was taken on a Lumar dissecting microscope (Zeiss) equipped with a CCD camera and the area covered by worm tracks was measured using Axiovision (Zeiss).Foraging behavior over 18 hrs was measured as the area of bacterial lawn that was explored by a single worm on 6 cm NGM plates seeded with TCA-precipitated proteins were urea-denatured, reduced, alkylated and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Roche) as described C. elegans Genome Arrays. Biotinylated cRNA was prepared from 300 ng Total RNA using the MessageAmp III kit according to the manufacturer instructions (Ambion). Data was analyzed using the R statistical environment. CEL files resulting from array analysis were interpreted and normalized using RMA Three independent populations of each strain were harvested at late L4/young adult stage. Total RNA was extracted using TRI reagent (Molecular Research Center) according to manufacturer's instructions. Microarray analysis was performed using Affymetrix GeneChip rpl-32 gene whose expression does not vary under our experimental conditions. Fold change was calculated using the Pfaffl method.At least two independent populations of each strain were harvested at late L4/young adult stage. Total RNA was extracted using TRI reagent (Molecular Research Center) according to manufacturer's instructions. Genomic DNA contamination was removed using the TURBO DNA-free kit (Ambion) and reverse transcribed using the RETROscript kit (Ambion). The cDNA was subjected to real time PCR analysis using the IQ SYBR Green supermix (Bio-Rad) on an iCycler (Bio-Rad). Each cDNA sample was amplified in triplicate reactions. The primers for Y45F10C.2 and the internal control rpl-32 were checked for specificity by direct sequencing of the PCR products and tested for efficiency with a dilution series of the template. All values were normalized against the Figure S1hjIs30[hsp::3xFLAG::EGL-4(K499A)::SL2::mCherry] at specified time after heat shock at 33∘C for 30 mins. Endogenous and FLAG-tagged EGL-4 protein was detected using anti-EGL-4 antibody. The α-tubulin blot served as loading control. (C) Expression level of EGL-4 in lysates prepared from transgenic animals carrying hjIs28[hsp::3xFLAG::EGL-4(K162N)::SL2::mCherry] at specified time after heat shock at 33∘C for 30 mins. Endogenous and FLAG-tagged EGL-4 protein was detected using anti-EGL-4 antibody. The α-tubulin blot served as loading control. (D) Foraging behavior of wild-type, hjIs28 and saeg-2(ok3174); hjIs28 animals that were not heat-shocked (nHS), 2 hrs after heatshock (HS-2hrs) or 24 hrs after heatshock (HS-24hrs). Quantitation of behavior was performed as in saeg-2;hjIs28 nHS (n = 6). . (E) Number of eggs retained in uterus in wild-type, hjIs28 and saeg-2(ok3174); hjIs28 animals that were not heat-shocked (nHS), 2 hrs after heatshock (HS-2hrs) or 24 hrs after heatshock (HS-24hrs). Total number of animals for each treatment of each strain: n = 20 .(A) Expression level of EGL-4 in lysates prepared from strains of indicated genotypes using anti-EGL-4 antibody. The antibody also recognized a non-specific protein (marked by an asterisk). The α-tubulin blot served as loading control. (B) Expression level of EGL-4 in lysates prepared from transgenic animals carrying (PDF)Click here for additional data file.Figure S2egl-4 mRNA level in strains of indicated genotypes, measured by real-time PCR. Results shown are derived from two independent mRNA samples for each strain assayed in triplicates. The mRNA level in wild-type (WT) animals was set as 1. (B) Expression level of EGL-4 in lysates prepared from strains of indicated genotypes using anti-EGL-4 antibody. The antibody also recognized a non-specific protein (marked by an asterisk). The α-tubulin blot served as loading control. (C) Expression level of SAEG-2 in lysates prepared from strains of indicated genotypes using anti-SAEG-2 antibody. The α-tubulin blot served as loading control. (D) SAEG-2::GFP nuclear localization is not affected by EGL-4 activity. hjSi15[saeg-2p::saeg-2::GFP-3xFLAG] single copy transgene was introduced into saeg-2(ok3174), egl-4(mg410); saeg-2(ok3174) and egl-4(n479); saeg-2(ok3174) mutant backgrounds. Confocal images of the head region centering on neurons at the nerve ring (marked by brackets) are shown.(A) The (PDF)Click here for additional data file.Figure S3egl-4(mg410) and egl-4(n479) animals using anti-EGL-4 antibody. Scale bar  =  10 mm.Immunostaining of dissected intestine of 1-day old adult wild type, (PDF)Click here for additional data file.Figure S4Drosophila S2 cells. (B) Co-immunoprecipitation of FLAG-tagged EGL-4 (FLAG-EGL-4) but not FLAG-tagged Venus (FLAG-Venus) with HA-tagged SAEG-2 (HA-SAEG-2) upon co-expression in Drosophila S2 cells. In the same experiment, co-immunoprecipitation of HA-tagged FLAG-EGL-4 with SAEG-1 (HA-SAEG-1) was reproduced. (C) Co-immunoprecipitation of FLAG-tagged PKG-Iβ(S64D) (FLAG-PKG) but not FLAG-tagged Venus (FLAG-Venus) with HA-tagged ZNF541 (HA-ZNF541) upon co-expression in HEK293 cells. (D) Co-immunoprecipitation of FLAG-tagged PKG-Iβ(S64D) (FLAG-PKG) and FLAG-tagged ZNF541 (FLAG-ZNF541) but not FLAG-tagged Venus (FLAG-Venus) with HA-tagged Dnttip1 (HA-Dnttip1) upon co-expression in HEK293 cells.(A) Co-immunoprecipitation of FLAG-tagged SAEG-2 (FLAG-SAEG-2) but not FLAG-tagged yellow fluorescent protein Venus (FLAG-Venus) with HA-tagged SAEG-2 (HA-SAEG-2) upon co-expression in (PDF)Click here for additional data file.Figure S5egl-4(n479) animals using anti-EGL-4 antibodies. (A–B) Nuclear staining was detected with anti-EGL-4 antibodies in a wild-type uterine epithelial cell (red arrowhead). (C–D) Nuclear staining was absent in an egl-4(n479) uterine epithelial cell (red arrowhead), demonstrating the specificity of the antibodies. Similar staining pattern was observed in at least 3 other samples for each genotype. Note background cytoplasmic staining in somatic gonadal cells in (A) and (C) but specific nuclear staining of the same cells in (A) (white arrowheads). Boxed areas in (A) and (C) were imaged at higher magnification and shown in (B) and (D). Scale bar  =  10 mm.Immunostaining of dissected uteri of 1-day old adult wild type and (PDF)Click here for additional data file.


The ATLANTIC-DIP (diabetes in pregnancy) programme showed 18% of women with gestational diabetes mellitus (GDM) screened with a 75g oral glucose tolerance test (OGTT) 12 weeks post-partum demonstrated glucose intolerance. However, long-term data on progression to type 2 diabetes (T2DM) post gestational diabetes (GDM) in an Irish population is lacking.We compared Caucasian women with previous GDM (n=116), and with normal glucose tolerance (NGT) during pregnancy (n=52), using a 75g OGTT, to determine prevalence of diabetes/pre-diabetes 1-5 years post index pregnancy. Women with abnormal OGTT 12 weeks post-partum  did not undergo OGTT, but were included in the analysis. American Diabetes Association diagnostic criteria for IFG/IGT/DM were used.Twelve percent (11/94) of GDM patients rescreened had pre-diabetes/DM , giving a prevalence of 28.4% (33/116) for pre-diabetes/diabetes, versus 2% (1/52) of women with NGT during pregnancy. Logistic regression analysis was used to determine index pregnancy factors associated with post–partum pre-diabetes/diabetes. These were: first-degree relative with DM , insulin use during pregnancy , fasting glucose during pregnancy . and not breastfeeding . BMI in pregnancy was not associated with pre-diabetes/diabetes at 1-5 years.The high prevalence of diabetes/pre-diabetes in this population offers an opportunity to develop a screening program to benefit at risk individuals, particularly targeting those with insulin-requiring GDM, higher fasting glucose levels and positive family history.


To compare signal to noise ratio (SNR) and contrast to noise ratio (CNR) for myocardium and blood during cardiac MR imaging in the newborn using 2 different receive-array coils. Paediatric Body/Cardiac (Paed-Body) and Flex-M coils were compared under a clinical investigation agreement with Philips.CMR assessments of cardiac function at 3.0 Tesla have been shown to provide more complete data with greater reproducibility than existing echocardiographic methods in newborns . Howeverblood-SImyocardium)/noise.Cardiac MRI scans from 38 newborns of corrected gestation 26-41 weeks were retrospectively analysed in 2 weight-matched groups. All scans were performed in a Philips 3T MRI Scanner. Cine images were acquired with resolution 1.5/1.5/5mm. In 19 infants a 2-channel Flex-M coil was placed above and below the chest; in 19 an 8-channel Paed-Body receive-array coil was placed around the infant. Myocardial and blood signal intensity (SI) were measured at the intraventricular septum and left ventricular cavity respectively, at end-diastole from the mid-ventricular level of a short axis stack. The Paed-Body coil produced significantly higher SNR in myocardium , SNR in blood (221.2vs139.9 p=0.003) and CNR (119.8vs83.5 p=0.03) (Figure The Paed-Body receive-array coil produces significant increases in SNR and CNR when performing cardiac MR imaging in newborn infants including those weighing less than 2000g.


Colorectal cancer (CRC) arises as a consequence of genetic events such as gene mutation and epigenetic alteration. The aim of this study was to identify new hypermethylated candidate genes and methylation-based therapeutic targets using vincristine in CRC.We analyzed the methylation status of 27,578 CpG sites spanning more than 14,000 genes in CRC tissues compared with adjacent normal tissues and normal colon tissues using Illumina bead chip array. Twenty-one hypermethylated genes and 18 CpG island methylator phenotype markers were selected as candidate genes. The methylation status of 39 genes was validated by quantitative methylation-specific polymerase chain reaction in CRC tissues, adjacent normal tissues, normal colon cells, and three CRC cell lines. Of these, 29 hypermethylated candidate genes were investigated using the demethylating effects of 5-aza-2′-deoxycytidine (5-aza-dC) and vincristine in CRC cells.AKR1B1, CHST10, ELOVL4, FLI1, SOX5, STK33, and ZNF304 was restored by treatment with 5-aza-dC and vincristine.Thirty-two out of 39 genes were hypermethylated in CRC tissues compared with adjacent normal tissues. Vincristine induced demethylation of methylated genes in CRC cells to the same extent as 5-aza-dC. The mRNA expression of AKR1B1, CHST10, ELOVL4, SOX5, STK33, and ZNF304 may be potential methylation biomarkers and therapeutic targets of vincristine in CRC.These results suggest that these novel hypermethylated genes  Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death in the world. CRC is a consequence of genetic events including gene mutations and epigenetic alterations that transform colonic epithelial cells into adenocarcinoma cells. The earCDKN2A, THBS, and SFRP, have been proposed to be associated with CRC tumorigenesis/[(GENE)M.SssI/(ACTB)M.SssI] × 100. The significance of different PMR values among CRC tissues and adjacent normal tissues was defined with the chi-squared test and analysis of variance test using Sigma Stat . All statistical tests were two-sided and P values of < 0.05 were considered to indicate statistical significance.The statistical significance of the methylation bead chip array data was determined using a paired t-test based on ΔP value of <0.001 based on a Δβ mean of 0.1. Among 3,177 CpG sites, we identified 597 genes with hypermethylated CpG sites in promoter CpG islands (data not shown). Finally, we selected 21 candidate genes that contained strongly hypermethylated CpG sites in promoter CpG islands in CRC tissues compared with adjacent normal tissues  and non-promoter regions , with aberrant methylated CpG sites identified in CRC tissues compared with adjacent normal and normal colon tissues, according to statistical significance determined by the paired t-test and an FDR FLI  and eight CIMP markers  were significantly demethylated by vincristine treatment in two more CRC cell lines . In our QMSP results of CIMP markers, DAPK1, TAC1, THBD, APC, CACNA1G, and CDKN2A were not significantly methylated in CRC tissues  family of transcription factors. It is well known that SOX5 regulates embryonic development and determines cell fate[STK33, serine-threonine kinase 33, is located on chromosome 11p15.3, a gene-rich region that has been associated with human diseases and malignancies[ZNF304, zinc finger protein 304, combines two conserved domains, class II AU-rich elements and a Krüppel-associated box, and is associated with the regulation of lymphocyte activation[The hypermethylation of c cancer,38. The ma cells. ELOVL4 ty acids. Hypermeanalysis,42. SOX5ell fate. STK33, gnancies. ZNF304,tivation.CHST10, ELOVL4, FLI1, STK33, SOX5, and ZNF304 in CRC cells. Interestingly, the methylation status of AKR1B1 was not affected, but its mRNA expression was increased by both drugs. It may be regulated by upstream genes, with a demethylating effect by both drugs. Our results provide insights into the potential functional impact of vincristine on methylated genes in CRC.DNA methylation-mediated silencing of gene expression can be restored by demethylation agents such as 5-aza-dC. DNA methyltransferase inhibitor, 5′-azacytidine, may act as an inducer of cell differentiation by causing de-methylation and re-expression of genes silenced by hypermethylation. 5-AzacyAKR1B1, CHST10, ELOVL4, SOX5, STK33, and ZNF304, and provided evidence for their suitability as methylation biomarkers of CRC. We also analyzed the DNA methylation-based therapeutic effects of vincristine in CRC.This study has identified novel candidate genes, ACTB: Beta-actin; ADAMTS1: A disintegrin and metalloproteinase with thrombospondin motifs 1; ADHFE1: Alcohol dehydrogenase, iron containing, 1; AKR1B1: Aldo-keto reductase family 1; APC: Antigen-presenting cell; BEND4: BEN domain containing 4; BTG4: B-cell translocation gene 4; CACNA1G: Calcium, voltage-dependent, T type, alpha 1G subunit; CDKN2A: Cyclin-dependent kinase inhibitor 2A; CHFR: Checkpoint with forkhead and ring finger domains; CHST10: Carbohydrate sulfotransferase 10; CIMP: CpG island methylator phenotype; CNRIP1: Cannabinoid receptor interacting protein 1; COP: Cyclophosphamide, vincristine and prednisone; CRABP1: Cellular retinoic acid-binding protein 1; CRC: Colorectal cancer; DAPK1: Death-associated protein kinase 1; ELOVL4: Elongation of very long chain fatty acids protein 4; EYA4: Eyes absent homolog 4; FDR: False discovery rate; FLI1: Friend leukemia integration 1 transcription factor; GLP1R: Glucagon-like peptide 1 receptor; IGFBP3: Insulin-like growth factor-binding protein 3; IGF2: Insulin-like growth factor 2; IRF4: Interferon regulatory factor 4; JAM2: Junctional adhesion molecule B; MGMT: O-6-methylguanine-DNA methyltransferase; MLH1: MutL homolog 1; M-MLV: Moloney murine leukemia virus; MTT: 3--2,5-diphenyltetrazolium; NEUROG1: Neurogenin-1; NPTX1: Neuronal pentraxin I; NRG1: Neuregulin 1; QKI: Quaking homolog, KH domain RNA binding; QMSP: Quantitative methylation-specific polymerase chain reaction; PMR: Percentage of methylated reference; RASSF1A: Ras association domain-containing protein 1; RT-PCR: Reverse transcription polymerase chain reaction; RUNX3: Runt-related transcription factor 3; SLC8A3: Solute carrier family 8, member 3; SFRP1: Secreted frizzled-related protein 1; SFRP2: Secreted frizzled-related protein 1; SOX5: SPY(sex determining region Y)-box 5; SPG20: Spastic paraplegia 20; STK33: Serine-threonine kinase 33; ST6GALNAC5: ST6 -N-acetylgalactosaminide alpha-2,6-sialyltransferase 5; TAC1: Protachykinin-1; THBD: Thrombomodulin; THBS: Thrombospondin; TWIST1: Twist-related protein 1; UNC5C: Unc-5 homolog C; VSX2: Visual system homeobox 2; WRN: Werner syndrome; ZNF272: Zinc finger protein 272; ZNF304: Zinc finger protein 304; 5-aza-dC: 5-aza-2′-deoxycytidine.The authors declare that they have no competing interests.JWM designed the study and drafted the manuscript. SKL and YWL prepared the clinical specimens and participated in the organization of clinical data. Dr. JOL, NK and SJK participated in the analysis of methylation array results and searching references. Dr. HJK helped the arrangement of results. Prof. JK provided the clinical specimens and clinicopathologic informations. Prof. HSK helped analyzed the results and revised the manuscript. Prof. SHP supervised the study and revised the manuscript. All authors read and approved the final manuscript.Table S2. Primers for mRNA expression.Primers for Quantitative methylation specific PCR (QMSP). Click here for file(A) and did not change significantly after treatment with concentrations of vincristine less than 100 nM for two or three days (B). *p-Values of <0.05 were considered statistically significant.Cell viability of CCD18Co, SW480, DLD-1, and LoVo cells by 5-aza-dC and vincristine. Cell viability of four cell lines after treatment with various concentrations of 5-aza-dC and vincristine for three days was evaluated by MTT assay. The cell viability of four cell lines was significantly reduced after treatment with concentration of 5-aza-dC greater than 30 μM Click here for file


If this is correct, then superoxide dismutase (SOD), which removes O2•−, should contribute to longevity assurance. In Caenorhabditis elegans, overexpression (OE) of the major cytosolic Cu/Zn-SOD, sod-1, increases life span. But is this increase caused by enhanced antioxidant defense? sod-1 OE did not reduce measures of lipid oxidation or glycation and actually increased levels of protein oxidation. The effect of sod-1 OE on life span was dependent on the DAF-16/FoxO transcription factor (TF) and, partially, on the heat shock TF HSF-1. Similarly, overexpression of sod-2  resulted in life-span extension that was daf-16 dependent. sod-1 OE increased steady-state hydrogen peroxide (H2O2) levels in vivo. However, co-overexpression of catalase did not suppress the life-span extension, arguing against H2O2 as a cause of longevity. sod-1 OE increased hsp-4 expression, suggesting increased endoplasmic reticulum (ER) stress. Moreover, longevity was partially suppressed by inactivation of ire-1 and xbp-1, mediators of the ER stress response. This suggests that high levels of SOD-1 protein may challenge protein-folding homeostasis, triggering a daf-16- and hsf-1-dependent stress response that extends life span. These findings imply that SOD overexpression increases C. elegans life span, not by removal of O2•−, but instead by activating longevity-promoting transcription factors.The superoxide free radical (O sod-1 over-expression increases levels of cellular ROS and of molecular damage. ► sod-1 over-expression effects on lifespan are dependent on DAF-16 (FoxO) and HSF-1. ► Increased lifespan is partially suppressed by inactivation of ER stress mediators. ► sod-2 (MnSOD) over-expression effects on lifespan are dependent on DAF-16.►  The oxidative damage theory suggests that SOD should protect against aging as well as oxidative damage. However, recent studies in Caenorhabditis elegans have found little support for this prediction 2•− generators actually increase life span C. elegans sod genes, abrogation of function has little or no effect on aging The oxidative damage theory proposes that accumulation of molecular damage caused by reactive oxygen species (ROS), particularly superoxide  slightly reduces life span sod-1 overexpression (OE) can increase life span sod-1 OE reduces levels of oxidative damage. Moreover, sod-1 OE lines were hypersensitive rather than resistant to oxidative stress, hinting that SOD OE might not reduce ROS-induced damage in these strains. It is also not known whether SOD levels in wild-type worms are limiting with respected to ROS detoxification.However, the effects of manipulation of expression of C. elegans, SOD OE can extend life span in Drosophila melanogaster2O2 levels in vivo prdx-2 leads to up-regulation of genes involved in phase II detoxification The effect on life span of transgene-induced elevation of SOD has been tested in several model organisms, with mixed results ; GA114, daf-16(mgDf50); daf-2(e1370); GA184, sod-2(gk257); GA222, wuEx118 [sod-2 gDNA rol-6(su1006)]; GA226, wuEx122 [sod-1 gDNA rol-6(su1006)]; GA228, wuEx123 [sod-1 gDNA rol-6(su1006)]; GA230, wuEx125 [sod-1 gDNA rol-6(su1006)]; GA644, wuEx123 [sod-1 gDNA rol-6(su1006)]; daf-16(mgDf50); GA646, wuIs152 [sod-1 gDNA rol-6(su1006)] hsf-1(sy441); GA648, wuIs152 [sod-1 gDNA rol-6(su1006)] aak-2(ok524); GA697, zcIs4 [hsp-4::gfp] wuEx196 [rol-6(su1006)]; GA800, wuIs151 ; GA801, wuIs152 [sod-1 gDNA rol-6(su1006)]; GA804, wuIs155 [sod-2 gDNA rol-6(su1006)]; GA805, wuIs156 [sod-2 gDNA rol-6(su1006)]; GA808, wuIs151 ; wuIs156 ; GA809, wuIs151 ; wuIs152 [sod-1 gDNA rol-6(su1006)]; GA812, wuIs156 [sod-2 gDNA rol-6(su1006)]; daf-16(mgDf50); GA824, wuEx196 [rol-6(su1006)]; GA1001, aak-2(ok524) (derived by outcrossing RB754); GA1407, zcIs4 [hsp-4::gfp] wuIs152 [sod-1 gDNA rol-6(su1006)]; GA1423, zcIs13 [hsp-6::gfp] wuIs152 [sod-1 gDNA rol-6(su1006)]; GA1424, zcIs13 [hsp-6::gfp] wuIs152 [sod-1 gDNA rol-6(su1006)]; GA1425, zcIs13 [hsp-6::gfp] wuIs152 [sod-1 gDNA rol-6(su1006)]; GA1429, zcIs13 [hsp-6::gfp] wuEx196 [rol-6(su1006)]; GA1430, zcIs13 [hsp-6::gfp] wuEx196 [rol-6(su1006)]; GR1307, daf-16(mgDf50); and PS3551, hsf-1(sy441). Standard nematode culture conditions were used Nematode strains used in this study included the following: DR1563, C. elegans lines overexpressing sod-2, a genomic DNA fragment containing the sod-2 coding sequence was amplified by PCR. This was injected into worms at 55 ng/μl, creating a line containing the wuEx118 transgene array. Primers used for PCR were 5′-TGAATCCTACGGAAAGTGCC-3′ and 5′-TCAATGAATGGACAGGTTTCCC-3′. wuIs155 and wuIs156 were created by integration of wuEx118 using X-irradiation. Integrant lines were outcrossed to N2 six times. Co-overexpression with catalase was achieved by combining the transgene arrays wuIs155 and wuIs151  in a single line. The construction of wuIs151 and of wuIs152 [sod-1 overexpression] has been described previously To prepare C. elegans hermaphrodites were grown at 20 °C and transferred to plates containing 10 μM fluorodeoxyuridine at the L4 stage of development. Death was scored as described previously Worms were transferred to control or NAC plates as L4/young adults and were transferred to freshly prepared plates every 7 days. NAC was added to plates topically from a freshly prepared NAC stock solution.2O2-specific fluorescent probe unc-119 mutant worms were transformed by biolistic transformation with a vector containing the wild-type unc-119 gene, the promoter of the constitutive and ubiquitously expressed rpl-17 gene, and the hyper coding region. Homozygous hyper transgenic worms were backcrossed twice into wild-type (N2). wuIs152 [sod-1] was then crossed into the hyper strain. Nematodes were cultured on NA agar seeded with Escherichia coli K12 as described previously hyper only (controls) or hyper plus sod-1 OE [wuIs152] were raised at 24 °C and harvested 1 day after the L3/L4 molt. Worms were washed with S-buffer  and next with 2.5 mM EDTA in S-buffer (to eliminate bacteria adhering to the cuticle) and pipetted as a dense pellet of at least 1000 worms in a black, flat-bottomed 96-microtiter plate well (Greiner). The fluorescent signal was measured over a 10-min period using a Victor2 1420 multilabel counter (PerkinElmer) at 25 °C with the excitation filters FP490 and FP405 and emission filter F535. The data shown represent the average ratios over 10 min of four biological replicates . A fuller description of the validation of this technique as a measure for standing H2O2 levels will be published soon by P. Back et al.HyPer is a genetically encoded HE. coli OP50. At harvest, late L4 larvae and young adult worms were rinsed off the plates, washed with M9 buffer, and stored at − 75 °C until use. For all assays, three to five replicate worm cultures were used.Synchronous cultures were initiated via alkaline hypochlorite lysis of gravid adults. Eggs were allowed to hatch overnight in M9 buffer at 20 °C and resulting L1 larvae were grown on NGM plates seeded with Worm samples were homogenized using a Bioruptor  in 2-ml microcentrifuge tubes containing equal amounts of suspended worms and CelLytic (Sigma) and 1× protease inhibitors (Roche). The resulting homogenate was centrifuged at 20,000 rpm for 30 min at 4 °C. The supernatant was collected, and cellular debris and intact worms were discarded. For phosphorylation assays, Phosphosafe buffer (Novagen) was used instead of CelLytic. Protein concentration of the supernatant was determined by the Bradford method (Bio-Rad).Thirty micrograms of each total protein extract was separated by SDS–PAGE and then electrotransferred onto a Hybond nitrocellulose membrane . Western blotting experiments were performed with anti-actin monoclonal antibodies (Santa Cruz Biotechnology) at a 1/1000 dilution, anti-Cu/Zn-SOD polyclonal antibody  at a 1/2000 dilution, anti-Mn-SOD polyclonal  at a 1/2000 dilution, anti-phospho-AMP-dependent protein kinase (AMPK) antibody  at a 1/1000 dilution, and anti-AGE monoclonal antibody (clone 6D12)  at a 1/1000 dilution. Detection of carbonyl groups was performed with the OxyBlot oxidized protein detection kit  according to the manufacturer's protocol. Samples (15 μg) of total protein extract were incubated for 15 min at room temperature with 2,4-dinitrophenylhydrazine to form the carbonyl derivative dinitrophenylhydrazone before SDS–PAGE separation. After transfer onto nitrocellulose, modified proteins were revealed by anti-dinitrophenol antibodies. Blots were developed with chemiluminescence using the SuperSignal West Pico chemiluminescent substrate (Perbio Sciences). Films were scanned and the amount of signal of each band or total lane (for carbonyl and carboxymethyllysine (CML)) was quantified by densitometric analysis using ImageQuant TL .SOD activity was measured using the Oxyselect superoxide dismutase activity assay  involving the inhibition of superoxide-induced chromogen chemiluminescence by SOD, according to the manufacturer's instructions. Total protein extracts (150 μg) were used for each measurement. For the measurement of Mn-SOD activity, protein extracts were pretreated with 5 mM NaCN to inhibit Cu/Zn-SOD activity. The absorbance of each well was read using a microplate reader  using 490 nm as the primary wavelength.t tests depending on the experimental circumstances.All results are expressed as the mean ± standard error of the mean. In cases in which several treatments or genotypes were compared to a single control, ANOVA was used for statistical analysis. In all other cases, we performed paired or unpaired sod-1 OE on three markers of molecular damage: protein carbonyl levels as a measure of irreversible protein damage sod-1 OE caused an ~ 1.5-fold increase in protein damage . The increased levels of protein oxidation might therefore reflect increased ROS levels.Elevated levels of protein carbonyls could be caused by an increase in protein oxidation or by a decrease in the turnover rate for oxidized proteins. To test the latter we measured the activity of the core (20S) proteasome, which is responsible for the degradation of oxidized proteins sod-1 OE does not increase life span by reducing ROS-mediated damage. An alternative possibility is that sod-1 OE triggers a protective, hormetic effect that increases life span. To explore this, we tested the dependence of the effects of sod-1 overexpression on three factors that can mediate responses to stress: AMPK and the transcription factors HSF-1 (heat shock factor) and DAF-16 (FoxO).The increased levels of protein oxidation, taken together with the reduced resistance to oxidative stress sod-1 OE robustly increased the life span of aak-2 mutants, which lack the major AMPK α subunit , and did not detectably increase AAK-2 phosphorylation levels . This argues against a role for AMPK in the effect of sod-1 OE on life span. By contrast, the magnitude of sod-1 OE life-span extension was markedly reduced in an hsf-1(sy441) background ) (hsf-1.daf-16(mgDf50) fully suppressed the longevity resulting from sod-1 OE  did not reduce either SOD-1 protein levels . The possible significance of this unexpected observation is considered in the Discussion.More strikingly, the null mutation OE lines D. These 2•− derived from mitochondrial respiration as a key contributor to aging. We therefore tested the effect on life span of overexpression of the major mitochondrial Mn-SOD isoform, sod-2C. elegans lines overexpressing sod-2 from transgene arrays, wuEx118, wuIs155, and wuIs156. wuIs155 and wuIs156 are integrated transgene arrays derived from wuEx118.The oxidative damage theory views Osod-2 OE extended mean and maximum life span by approximately 25% . We verified that elevated sod-2 gene copy number increased SOD-2 protein levels using Western blots . As for sod-1 OE, the life-span increase by sod-2 OE was daf-16-dependent . This revealed a modest but statistically significant increase in steady-state H2O2 levels in sod-1 OE animals .Because SOD activity generates H animals A. Amplexsod-1 overexpressing animals appeared unaffected by catalase co-overexpression (co-OE) 2O2 in inducing the DAF-16-dependent life-span extension. We reinvestigated this question and again found that catalase co-OE did not suppress longevity in two sod-1 OE strains . Furthermore, the increase in protein carbonylation in SOD-1 OE animals was not reduced to wild-type levels by catalase co-OE .However, we previously noted that the slight life-span increase of se co-OE C. We consod-1 OE longevity. To probe this, we used NAC (1 mM), which can act as a broad-spectrum antioxidant, but again sod-1 OE longevity was not suppressed . Potentially, this large increase in protein levels could upset protein folding homeostasis, e.g., by increasing levels of unfolded proteins in the endoplasmic reticulum (ER). Such ER stress can induce an unfolded protein response (UPR) to restore protein folding homeostasis. Expression of hsp-4, encoding an ER chaperone protein, is induced under conditions of ER stress sod-1 OE increased expression of hsp-4::gfp . Induction of the UPR can also increase thermotolerance, but we detected no difference between sod-1 OE and wild-type worms in resistance to heat stress (35 °C) (data not shown).Cu/Zn-SOD is a highly expressed cytosolic protein p-4::gfp C and D, C. elegans, IRE-1, PEK-1, and ATF-6 can sense ER stress and activate a UPR ire-1 and xbp-1 markedly reduced hsp-4::gfp expression in both the presence and the absence of sod-1 OE , whereas that of pek-1 and atf-6 did not (data not shown). ire-1 and xbp-1 RNAi did not fully abrogate the induction of hsp-4::gfp expression by sod-1 OE, but reduced its magnitude in terms of fold change increase . This could reflect either incomplete knockdown of ire-1 and xbp-1 by RNAi or a non-ire-1/xbp-1-dependent component of the effects of sod-1 OE on hsp-4::gfp expression. Overall, these results suggest that sod-1 OE induces an ER stress response. RNAi of xbp-1 and ire-1 also reduced the life span of sod-1 OE animals more than that of wild-type animals in two independent trials . This result is consistent with a role for the UPR in mediating the life-span extension by sod-1 OE.In sod-1 cytosolic Cu/Zn-SOD increases C. elegans life span and proposed that this was due to reduced levels of cytosolic O2•−sod-1 OE reduces resistance to oxidative stress 2O2, and extends life span only in the presence of the DAF-16/FoxO transcription factor. This is consistent with earlier observations that enhancing SOD activity in vivo by administration of SOD mimetics does not extend C. elegans life span Drosophila causes changes in gene expression that are similar to those found in C. elegans insulin/IGF-1 signaling mutants Drosophila life-span extension has not been tested, it seems possible that life-span extension involves mechanisms similar to those in C. elegans.We previously reported that overexpression of the Our results illustrate how overexpressing antioxidant genes can produce complex effects and have unexpected consequences, and emphasize the importance of interpreting such studies with care sod-1 OE might increase life span. First, we showed that sod-1 OE increases steady-state levels of H2O2 in vivo. However, results of catalase co-overexpression tests did not support the view that elevated H2O2 levels increase life span.We explored alternative mechanisms by which sod-1 might lead to levels of SOD-1 protein so high as to overwhelm the cellular protein folding machinery, inducing a UPR. Consistent with this, sod-1 OE increased expression of hsp-4::gfp and hsp-6::gfp, markers of the cytosolic and mitochondrial UPR, respectively. The increase in hsp-4::gfp expression was partially suppressed by RNAi of ire-1 or xbp-1, which also partially suppressed the longevity of sod-1 OE worms. This is consistent with the involvement of an ER stress response in the longevity of sod-1 OE worms. ire-1 and xbp-1 have previously been shown to promote longevity: mutation of either gene reduces life span in a daf-2(e1370) mutant sod-1 OE might increase life span by triggering a UPR.Expression studies imply that SOD-1 is an abundant cytosolic protein sod-1 and sod-2 requires the presence of daf-16, which is also required for the longevity of mutants with reduced IIS sod OE strains and long-lived IIS mutants, i.e., that similar daf-16-dependent changes in gene expression are operative. However, certain observations suggest that this might not be so. sod-3 is a direct transcriptional target of DAF-16 that shows daf-16-dependent up-regulation in daf-2 mutants sod-3 or mtl-1 (another daf-16 target) sod-1 OE lines sod-1 OE also proved to be daf-16 dependent . We also confirmed that protein oxidation levels are not increased in daf-2 mutants or, in daf-2 mutants, reduced by mutation of daf-16 , consistent with a previous study sod-1 OE and mutation of daf-2, DAF-16 might act in a different way to increase life span.The longevity induced by overexpression of daf-16-dependent increase in protein levels in sod-1 OE worms also implies that, in this context at least, DAF-16 does not increase life span by reducing levels of protein oxidation. Moreover, it provides further evidence that increasing levels of protein oxidation does not necessarily accelerate aging. Likewise, deletion of the sod-2 Mn-SOD increases oxidative damage levels but does not reduce life span The sod-1 and sod-2 overexpression is not a consequence of lowered levels of ROS or of oxidative damage. This means that the increased life span of these strains may not be taken as evidence in support of the oxidative damage theory of aging. In fact, the association of increased levels of protein oxidation with increased life span argues against the theory. The exact mechanism by which sod overexpression triggers a daf-16-dependent increase in life span remains unclear. However, our results suggest that high levels of SOD protein may trigger an unfolded protein response that contributes to the increase in life span.The results presented here imply that the increased life span caused by


Ligation of the TNF receptor family co-stimulatory molecule OX40 (CD134) with an agonist anti-OX40 mAb enhances anti-tumor immunity by augmenting T cell differentiation as well as turning off the suppressive activity of FoxP3+CD4+ regulatory T cells (Treg). In addition, antibody-mediated blockade of the checkpoint inhibitor, CTLA-4, releases the “brakes” on T cells to augment tumor immunotherapy. However, monotherapy with these agents can have limited therapeutic benefit particularly against poorly immunogenic murine tumors. Therefore, we examined whether the combination of agonist anti-OX40 therapy in the presence of CTLA-4 blockade would enhance tumor immunotherapy. Combined anti-OX40/anti-CTLA-4 immunotherapy significantly enhanced tumor regression and the survival of tumor-bearing hosts in a CD4 and CD8 T cell-dependent manner. Mechanistic studies revealed that combination immunotherapy directed the expansion of effector T-bethigh/Eomeshigh granzyme B+ CD8 T cells. Dual immunotherapy also induced among distinct populations of Th1  and, surprisingly, Th2  CD4 T cells exhibiting increased T-bet and Gata-3 expression. Furthermore, IL-4 blockade inhibited the Th2 response, while maintaining Th1 CD4 and effector CD8 T cells that enhanced tumor-free survival. These data demonstrate that refining the global T cell response during combination immunotherapy can further enhance the therapeutic efficacy of these agents.


Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC)  Spinocerebellar ataxias (SCAs) are a group of inherited neurodegenerative diseases with around 30 subtypes, which are characterized by a progressive loss of cerebellar neurons. Neuronal death has been linked to the aggregation of mutated disease-causing proteins, such as ataxin-1 (ATXN1). Pathogenic ATXN1 contains an elongated glutamine stretch, which triggers spontaneous misfolding and self-assembly of the protein into aggregates. Earlier studies in lower organisms have discovered many non-human proteins that alter aggregation and/or toxicity of mutant ATXN1. Here, we combine an experimental screening approach with bioinformatics to find human proteins that modulate aggregation and toxicity of ATXN1. We identified 21 proteins affecting mutant ATXN1 in mammalian cells. Further characterization revealed that enhancers of ATXN1-mediated toxicity contain α-helical coiled-coil domains as structural motifs, while suppressors do not. Detailed studies with the ATXN1 interacting proteins MED15 and Pum1 finally demonstrated that coiled-coil domains are indeed critical for the aggregation and toxicity promoting effects of human proteins. Our study contributes to a deeper understanding of ATXN1 aggregation and SCA1 pathogenesis and highlights potential therapeutic targets for further investigations. Accumulation of misfolded proteins with pathogenic polyglutamine (polyQ) tracts in insoluble aggregates is a hallmark of late-onset neurodegenerative diseases, including Huntington's disease (HD) and spinocerebellar ataxias (SCAs) Various experimental studies indicate that pathogenic polyQ tracts with ∼40 or more glutamine residues drive spontaneous misfolding and aggregation of disease proteins such as huntingtin (HTT), ataxin-1 (ATXN1) and ataxin-3 (ATXN3) The kinetics of spontaneous polyQ-mediated protein assembly is influenced by the concentration, the amino acid composition and the size of a disease protein. For instance, full-length HTT, with a pathogenic polyQ tract (≥40 glutamines), does not spontaneously self-assemble into insoluble protein aggregates in mammalian cells In the last decade a number of proteins have been reported to influence misfolding and aggregation of polyQ-containing disease proteins in model systems like yeast, worm or flies in vitro, suggesting that the N-terminal CC is critical for this aggregation enhancing effect. Further studies with recombinant proteins demonstrated that the N-terminal CC of MED15 (MED15CC) alone is sufficient to promote spontaneous ATXN1 aggregation in cell-free assays. We also observed that a hybrid Pum1-MED15CC fusion protein potently enhances ATXN1 aggregation in mammalian cells, while such an effect was not observed with the wild-type Pum1 protein. Finally, cell-based assays revealed that a truncated MED15 fragment lacking the N-terminal CC (MED15ΔCC) in contrast to the wild-type protein does not promote spontaneous ATXN1 aggregation. Together, this supports the hypothesis that CC domains in interacting proteins are critical for stimulation of spontaneous ATXN1 aggregation. The potential implications of these results for the pathogenesis of polyQ disorders and the development of novel therapeutic strategies are discussed.In this study, we searched for human proteins that influence toxicity and aggregation of pathogenic ATXN1 in cell model systems. Using literature information on lower model organisms we first predicted ∼200 potential human modifiers. These were then systematically tested in cell-based assays. Using this approach, we identified 21 human proteins, most of which significantly influenced both toxicity and aggregation of pathogenic ATXN1 in mammalian cells. In computational studies we predicted that many ATXN1 toxicity enhancers contain putative coiled-coil (CC) domains. Strikingly, no such domains were predicted for toxicity suppressors, suggesting that CC regions in modifiers are critical for their aggregation and toxicity promoting effects on polyQ disease proteins. We then investigated in more detail whether the ATXN1 interacting proteins MED15 and Pum1 directly influence polyQ-mediated ATXN1 aggregation in cell-free assays. MED15 contains a relatively large computationally predicted N-terminal CC domain, while such a domain was not predicted for Pum1. We found that MED15 but not Pum1 efficiently promotes spontaneous ATXN1 aggregation Caenorhabditis elegansDrosophila melanogasterSaccharomyces cerevisiaeMus musculusPreviously, proteins that modify the toxicity and aggregation of different polyQ-containing disease proteins have been identified using various model organisms, including For expression analysis, 25 plasmids were randomly selected and transfected into COS-1 cells. Synthesis of N-terminally FLAG-tagged recombinant fusion proteins was verified by SDS-PAGE and immunoblotting. We verified 22 of 25 randomly selected plasmids (88%) to express FLAG-tagged proteins at approximately the expected size in mammalian cells , suggestNT and ATXN1Q82NT) were constructed and transiently transfected into COS-1 cells. After 48 h toxicity was monitored using a standard caspase-3/7 assay. We observed that caspase-3/7 activity was ∼2-fold higher in cells with pathogenic YFP-ATXN1Q82NT than in cells with non-pathogenic YFP-ATXN1Q30NT or the control protein YFP  and insoluble protein fractions (pellet) were prepared from crude COS-1 cell extracts by centrifugation and analyzed by SDS-PAGE and immunoblotting . We founng cells  is not cng cells , indicatNT toxicity modifiers in cell-based assays is schematically shown in NT and one of the 202 selected modulator proteins. After 48 h caspase-3/7 activity was quantified in cell extracts. Modulators that enhanced or suppressed YFP-ATXN1Q82NT toxicity compared to the control plasmid pFLAG-GW in three independent experiments by at least 20% were selected. Hits from primary screens were systematically re-tested in a control toxicity assay, monitoring the effect of modulator proteins on caspase-3/7 activity in the absence of YFP-ATXN1Q82NT  of 21 tested proteins also significantly influence YFP-ATXN1Q82NT aggregation in cell-based assays  caused a reduction of YFP-ATXN1Q82NT toxicity but at the same time increased YFP-ATXN1Q82NT aggregation  was established (NT toxicity modulators (19%) influenced YFP-ATXN3Q73 toxicity in cell-based assays . We systematically tested 15 selected siRNAs in cell-based assays, 10 of which (67%) reproducibly influenced the toxicity of YFP-ATXN1Q82NT  in the well-established SCA1 transgenic fly model d assays . We systd assays , as prevd assays , confirmd assays . The oppd assays , suggestNT toxicity modulators using the EASE program NT toxicity modulators  domains are critical for the spontaneous aggregation and toxicity of polyQ disease proteins in vitro and in vivoPrevious studies have also demonstrated that proteins with short, non-pathogenic polyQ tracts bind to neurodegenerative disease proteins with long, pathogenic polyQ tracts and influence their aggregation propensity NT toxicity in cell-based assays  spectroscopy in vitro. For comparison, we observed a [θ]222/[θ]208 ratio of 0.84 for the control protein GST whose alpha-helical structure is known to lack coiled coils Our data with cell-free and cell-based aggregation assays suggest that the conserved N-terminal CC domain in MED15  is critiin vitro.Finally, we investigated whether MED15CC directly influences spontaneous ATXN1Q82 aggregation in cell-free assays. GST-ATXN1Q82 fusion protein (2 µM) was incubated with PP and GST-MED15CC (2 or 7 µM); the formation of SDS-stable ATXN1Q82 aggregates was quantified after 24 and 48 h by filter retardation assay . We founOur data indicate that MED15CC alone is sufficient to promote spontaneous ATXN1Q82 aggregation in cell-free assays . This suNT aggregation were investigated . In contrast to YFP-ATXN1Q82an cells . We founan cells .NT by confocal microscopy  influences spontaneous ATXN1 aggregation . The mChNext, we used the established COS-1 cell model to investigate the effects of MED15 and MED15ΔCC on YFP-ATXN1Q82 toxicity. FLAG-tagged MED15 and MED15ΔCC fusion proteins were co-produced with YFP-ATXN1Q82 in COS-1 cells and after 48 h the caspase-3/7 activity was quantified in cell extracts. We found that YFP-ATXN1Q82 toxicity was significantly increased in the presence of full-length MED15 protein but not in the presence of the truncated fragment MED15ΔCC , indicatFinally, we examined whether the N-terminal CC domain in MED15 influences the association of the protein with YFP-ATXN1Q82 in mammalian cells using confocal microscopy . We obseIn previous studies a large number of modifier proteins have been identified that influence misfolding or toxicity of polyQ disease proteins such as huntingtin, ataxin-1 or ataxin-3 NT) in cell-based assays. Our investigations revealed 21 modulators that reproducibly enhanced or reduced the toxicity of YFP-ATXN1Q82NT , which mediates tRNAmet binding to ribosomes and controls global protein synthesis NT might be due to a re-activation of eIF2 function, leading to improved protein translation and reduced apoptosis.Proteins involved in translation were also overrepresented among ATXN1 toxicity modifiers in this study  domains are critical for the spontaneous aggregation of Q/N-rich yeast prions and polyQ disease proteins  domains . In addi domains , suggest domains , while s domains . However domains . In vitrC domain .in vitro, supporting the hypothesis that this N-terminal domain is critical for MED15-mediated stimulation of ATXN1 aggregation  adopts a typical α-helical CC structure . Moreoveregation . We alsoregation . Finallyregation . Thus, oSeveral lines of experimental evidence indicate that long polyQ tracts in neurodegenerative disease proteins such as ATXN1 have to undergo a conformational change before they can assemble into insoluble protein aggregates D. melanogaster or C. elegans are necessary to elucidate the cellular processes that increase or reduce the toxicity of pathogenic polyQ proteins. We suggest that a step-by-step process of data integration and refinement as well as dissection of molecular details in signaling cascades and cellular protein quality control pathways will lead to the discovery of new drug targets and therapeutic strategies, which can be tested in appropriate disease models and patients with polyQ diseases.Taken as a whole, our study shows that a strategy combining bioinformatic and experimental approaches is useful for the identification of human modifier proteins that influence the toxicity and aggregation of pathogenic ATXN1. These modifiers play a functional role in a variety of different cellular mechanisms that are currently not very well understood. Further investigations with human proteins and their homologues from lower model organisms such as NT (458 aa), ATXN1Q82NT (508 aa), ATXN3Q25 (360 aa) and ATXN3Q73 (408 aa) were PCR amplified with Pwo SuperYield DNA polymerase (Roche) from the plasmids pOTB7-MED15 (cloneID:CCSB7005), pAS2-1-ATAXIN1Q30, pAS2-1-ATAXIN1Q82, pBTM116-ATXN3Q25 and pBTM116-ATXN3Q73 and cloned into the Gateway plasmid pDONR221 (Invitrogen). Oligonucleotides  at a 1∶2 ratio in the presence of BP clonase (Invitrogen). After incubation for 16 h at 25°C the resulting entry DNA plasmids were transformed into competent Mach1 E. coli cells (Invitrogen). E. coli clones were selected on LB agar plates supplemented with Kanamycin and analyzed by BsrGI restriction digestion (NEB). Finally, the identity of DNA fragments was verified by DNA sequencing. The pJET-MED15CC plasmid was generated by isolating the 554 bp PCR product from an agarose gel and blunt end cloning into the pJET1.2 vector (Fermentas) according to the manufacturer's recommendations. Identity of the DNA fragment was verified by DNA sequencing. To create the Gateway compatible entry plasmid encoding Pum1-MED15CC (pePUM1-MED15CC), the 537 bp cDNA fragment MED15CC was subcloned into the unique AatII site in the Gateway compatible plasmid pOBT7-Pum1 (cloneID:CCSB6127) encoding the Pum1 protein , ATXN1Q30 (818 aa), ATXN1Q82 (868 aa), ATXN1Q30 protein . The cor protein . For ampNT was cloned into the pdEYFP-C1Amp vector (Imagenes). cDNA fragments encoding ATXN1Q30 and ATXN1Q82 were cloned into the vector pPA-Reni-DM E. coli, the cDNA fragments encoding ATXN1Q82 and MED15CC were cloned into the expression vector pGEX-6P-1 (Amersham Biosciences); cDNA fragments encoding the proteins MED15 and Pum1 were subcloned into the plasmid pDESTCo (Invitrogen). For generation of destination plasmids the Gateway recombination cloning technology was applied as recommended by the manufacturer (Invitrogen). Entry plasmids and empty destination vectors (E. coli cells (Invitrogen) as recommended by the manufacturer. Finally, plasmid DNAs were purified from E. coli clones using the QIAprep 96 Turbo Miniprep Kit (Qiagen) and analyzed by BsrGI (NEB) restriction digestion.For cell-based modifier screens, we cloned 202 computationally selected genes  into the vectors  were comNT and the FLAG-tagged modifier protein MED15. Transfections were performed using Lipofectamine 2,000 in Opti-MEM (Invitrogen). In each co-transfection experiment ∼200 putative modifiers were systematically tested. All co-transfection experiments were performed in triplicates. Transfected COS-1 cells were harvested after 48 h and lysed in 1% NP-40 PBS, supplemented with a complete protease inhibitor cocktail mix (Sigma). To separate the supernatant from the insoluble pellet, crude cell extracts were centrifuged at 30,000 rpm for 30 min. The pellet was dissolved in 8 M urea.COS-1 cells were grown to 70–80% confluency in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal calf serum, penicillin (100 µg/ml) and streptomycin (100 µg/ml). For cDNA overexpression experiments, cells at passage 5–6 were seeded into 96-well cell culture plates . Then, they were co-transfected with pdEYFP-C1Amp (0.4 µg) and a pFLAG-GW-based plasmid (0.4 µg) encoding e.g., the proteins YFP-ATXN1Q82NT. The mouse monoclonal anti-FLAG antibody M2 (Sigma-Aldrich) was used  for the detection of FLAG-tagged modifier proteins produced in COS-1 cells. Recombinant ATXN3Q25 and ATXN3Q73 proteins were detected with the anti-MJD CT antibody  To generate a polyclonal antibody against ATXN1, two peptides  were independently fused to the carrier Keyhole Limpet Hemocyanin (KLH) protein and injected into rabbits using standard immunization procedures . The resulting immune sera were affinity purified against His-tagged ATXN1Q30 immobilized on a Ni-NTA column (Qiagen), according to manufacturer's instructions. The specificity of antibodies was confirmed by SDS-PAGE and immunoblotting using protein extracts from transiently transfected COS-1 cells . The antNT and 2 pmol target gene siRNA in 24 well plates . siRNAs were obtained from the HP GenomeWide siRNA library (Qiagen). The cell culture conditions and transfection reagents were as described in protein overproduction experiments. To confirm the knock-down of target genes qRT-PCR experiments were performed. RNA was purified from cells using the RNeasy Mini Kit (Qiagen) as recommended by the manufacturer. cDNA generation and qRT-PCR reactions were performed using the Dynamo FLASH SYBR Green qPCR kit (Finnzymes) in an Applied Biosystems 7,500 operating system. Each experiment was performed in triplicate. The sequences of oligonucleotides used in qPCR experiments are displayed in For siRNA assays, COS-1 cells were transfected with 0.2 µg of a pdEYFP-C1Amp vector encoding YFP-ATXN1Q82NT and a potential FLAG-tagged modifier protein. Caspase 3/7 activity in transfected COS-1 cells was measured using the Apo-ONE Homogeneous Caspase-3/7 Assay kit (Promega). Briefly, cell lysis buffer was mixed with a pre-fluorescent caspase 3/7 substrate and added to COS-1 cells 48 h after transfection. The pre-fluorescent caspase 3/7 substrate was cleaved by activated caspase 3/7 and resulted in the formation of a fluorescent product. Fluorescence was measured in a plate reader (TECAN Infinite M200) and caspase 3/7 activity was normalized to the cell numbers quantified via high content imaging microscopy . Each experiment was performed in triplicates.An equal number of COS-1 cells  were co-transfected with plasmids encoding YFP-ATXN1Q82For quantification of insoluble polyQ-containing protein aggregates a filter retardation assay was applied 2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g/l D-glucose, 10% fetal calf serum, penicillin (100 µg/ml) and streptomycin (100 µg/ml). Cells were seeded in 96-well cell culture plates  and co-transfected with a 1∶1 mix of cDNAs encoding YFP-ATXN1Q82 or YFP-ATXN1Q82NT and mCherry-tagged modifiers (200 ng each). 48 h after transfection cells were fixed with 4% paraformaldehyde (Sigma) and DNA was stained with Hoechst 33342  (Invitrogen). The cell culture plates were then placed into a high-content screening (HCS) cell analysis system . The principle of HCS analysis is described elsewhere NT and ATXN3 overproduction was quantified by YFP fluorescence and modifiers were monitored by mCherry fluorescence. The fluorescence signals were normalized to cell numbers. Experiments were performed in triplicate.SH-EP cells were grown at 37°C and 5% CO2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g/l D-glucose, 10% fetal calf serum, penicillin (100 µg/ml) and streptomycin (100 µg/ml). Cells were seeded on coverslips in 24-well cell culture plates . Co-transfections were performed using cDNAs encoding YFP-ATXN1Q82, YFP-ATXN1Q82NT and mCherry-tagged modifiers (500 ng each). For single transfections cells were transfected with cDNAs encoding YFP-ATXN1Q30, YFP-ATXN1Q82, YFP-ATXN1Q30NT or YFP-ATXN1Q82NT (500 ng each). 48 h after transfection cells were fixed with 4% paraformaldehyde (Sigma), stained with Hoechst 33342  and mounted using ProLong Gold antifade reagent (Invitrogen). ATXN1Q82 and ATXN1Q82NT overproduction was monitored by YFP fluorescence and modifiers were detected by mCherry fluorescence. Images were recorded using a Leica SP2 confocal microscope.COS-1 cells were grown at 37°C and 5% COhttp://stockcenter.vdrc.at/control/main). UAS-GFP and UAS-GFP-RNAi lines, provided by C. Antoniewski were used as controls. J. Botas and M. O. Fauvarque kindly provided the fly lines harbouring UAS-ATXN1Q82 and GMR-GAL4 genes. Initially, a stable UAS-ATXN1Q82;GMR-GAL4 line was generated. To analyze the effects of niki and MED15 target gene knock-downs on ATXN1Q82 induced eye degeneration UAS-ATXN1Q82;GMR-GAL4 females were crossed with males of niki and MED15 RNAi lines and with males of UAS-GFP and UAS-GFP-RNAi control lines, respectively. The eye structure of male progeny (n>20) was examined using Leica and Zeiss stereomicroscopes. For electron microscopy imaging, flies (n>5) were desiccated at 40°C overnight, dried by the critical point method with liquid N2, sputter-coated with gold and visualized with a scanning electron microscope (JEOL JSM 840-A).For knock-down of niki and MED15 gene expression the VDRC stocks 16,120 and 21,809 were used (For LUMIER assays protein A (PA)-Renilla luciferase (RL)-tagged ATXN1 fusion proteins (PA-RL-ATXN1Q30 or PA-RL-ATXN1Q82) were co-produced with firefly luciferase-tagged modulator proteins FL-Pum1, FL-MED15 or FL-U2AF2 in HEK293 cells. After 48 h protein complexes were co-immunoprecipitated from cell extracts with IgG coated magnetic beads ; interactions between bait (PA-RL fusions) and prey proteins (FL fusions) were monitored by quantification of firefly luciferase activities E. coli BL21 cells. Recombinant proteins were purified using glutathione agarose (Sigma) or Ni-NTA agarose (Qiagen) beads according to the instructions of manufacturers. His-tagged Pum1 protein was purified under denaturing conditions using 6 M guanidium-HCl and refolded in buffer A (500 mM (NH4)2SO4, 50 mM MgCl2, 10 mM DTT, 30% glycerol) for 16 h on ice. The supernatant was dialyzed against buffer B  for 16 h at 4°C. His-Pum1 protein was then concentrated using mini spin-columns (Millipore).For the production of GST- and His-tagged fusion proteins the genes encoding the proteins ATXN1Q82, MED15CC, MED15 and Pum1 were cloned into the vectors pGEX-6P-1 and pDEST-Co and the resulting plasmids pGEX-6P-1-ATXN1Q82, pGEX-6P-1-MED15CC, pDEST-Co-MED15 and pDEST-Co-Pum1 were transformed into in vitro aggregation assays, GST-ATXN1Q82 (5–7 µM) was incubated with His-tagged MED15, His-tagged Pum1 or GST-tagged MED15CC in the presence of PreScission Protease (Amersham Pharmacia) at 30°C with constant shaking (300 rpm). Reactions were stopped by freezing of samples in liquid nitrogen. 25 µg of GST-ATXN1Q82 was filtered through a 0.2 µm cellulose acetate membrane and protein aggregates retained on filter membranes were detected using the polyclonal anti-ATXN1 antibody SA4645 .For 2HPO4/NaH2PO4, 150 mM NaF pH 7.5 for 16 h at 4°C. Far-UV circular dichroism spectra of GST and GST-MED15CC (5 µM) were recorded at 20°C using a Chirascan CD spectrometer (Applied Biophysics) at a bandwidth of 1 nm. Spectra were collected at 1 nm intervals from 250 to 190 nm and at least 3 average values for each measurement were smoothed and normalized to mean residual weight ellipticity [θ] using the Pro-Data software (Applied Biophysics). MED15CC CD values were acquired by substraction of the GST CD spectrum from the GST-MED15CC CD spectrum. Prediction of secondary protein structure based on the CD data was performed using the K2D2 program GST and GST-MED15CC proteins were dialyzed in 50 mM NaNT toxicity modifiers. The modifier genes were compared to the human genome; a Fisher's exact test was performed to identify over-represented functional categories. We only considered categories with adjusted p-values<0.05 (adjusted with the Bonferroni method) as statistically significant. For the prediction of coiled-coil domains (CC) in amino acid sequences of modifier proteins the COILS program was used NT toxicity modifiers with CC domains to the human proteome .The EASE program To identify glutamine-rich (Q-rich) or polyglutamine (polyQ) proteins, a window of 50 amino acids was passed over each protein sequence. For each window position the fraction of Qs amongst all residues or the number of consecutive Qs was counted. A protein was considered as Q-rich when it contains >50% Qs in a stretch of at least 10 amino acids; in comparison a protein was defined as a polyQ protein when it contains a stretch of >10 consecutive Qs.Sequence homologues of human Pum1 and MED15 were selected using the NCBI BLAST sequence search program Figure S1Western blot analysis of FLAG-tagged modulator proteins. Plasmids encoding FLAG-tagged modifier proteins were randomly selected and transfected into COS-1 cells. Protein extracts were prepared after 48 h and analyzed by SDS-PAGE and immunoblotting using the anti-FLAG antibody M2. The same amount of total protein was loaded in each lane.(TIF)Click here for additional data file.Figure S2NT or YFP-ATXN1Q82NT aggregates in COS-1 cells. (A) A schematic representation of full-length and truncated ATXN1 proteins with pathogenic and non-pathogenic polyQ tracts. (B) Confocal microscopy pictures of YFP-ATXN1Q82NT and YFP-ATXN1Q30NT protein aggregates detected in COS-1 cells. Cells were analyzed after 48 h. Nuclei are stained with Hoechst 33342 (blue) and YFP-ATXN1Q82NT and YFP-ATXN1Q30NT protein deposits are shown in green. (C) Quantification of YFP-ATXN1Q30NT and YFP-ATXN1Q82NT aggregates in COS-1 cells using a high-content screening cell analysis system .Detection and quantification of YFP-ATXN1Q30(TIF)Click here for additional data file.Figure S3NT and a potential toxicity modifier. Human proteins that either enhance (green) or suppress (red) the toxicity of pathogenic YFP-ATXN1Q82NT are identified in a cell-based screen and subsequently retested in cells in the absence YFP-ATXN1Q82NT fusion protein. Modulators were only considered for further investigations when they specifically influence YFP-ATXN1Q82NT cytotoxicity in cell-based assays.Schematic overview of modifier cytotoxicity screening strategy. COS-1 cells were co-transfected with pairs of plasmids encoding YFP-ATXN1Q82(TIF)Click here for additional data file.Figure S4NT toxicity modifiers in COS-1 cells. Overproduction of ATXN1Q82NT cytotoxicity modifiers in transiently transfected COS-1 cells was analyzed after 48 h by SDS-PAGE and immunoblotting using the anti-FLAG antibody M2. Asterisks indicate modifier proteins.Western blot analysis confirms the production of YFP-ATXNQ82(TIF)Click here for additional data file.Figure S5NT toxicity modifiers (Effects of human proteins on pathogenic ATXN3 cytotoxicity and aggregation. (A) Relative caspase 3/7 activity change induced by overproduction of proteins YFP-ATXN3Q25 or YFP-ATXN3Q73 in COS-1 cells compared to YFP overproducing cells. Caspase 3/7 activity was significantly increased in cells producing the ATXN3 fusion protein with a pathogenic polyQ tract compared to the wild-type protein . Error bars indicate SD. (B) Detection of SDS insoluble protein aggregates by filter retardation assay. The YFP-ATXN3Q73 fusion protein forms SDS-insoluble protein aggregates. Equal amounts of total protein were loaded. (C) Effects of modifier proteins on YFP-ATXN3Q73 cytotoxicity; 21 previously identified YFP-ATXN1Q82odifiers  were sys(TIF)Click here for additional data file.Figure S6siRNA treatment reduces the expression of modifier genes in mammalian cells. siRNA treated COS-1 cells were analyzed by qRT-PCR. siRNA knock-down of six randomly selected target genes caused a reduction of mRNA levels by 50–90% in comparison to the non-targeting control. mRNA levels were normalized to the mRNA levels of β-actin. Data is shown as mean ± SD for three independent experiments performed in triplicates.(TIF)Click here for additional data file.Figure S7Homo sapiens; mm, Mus musculus; dm, Drosophila melanogaster; ce, Caenorhabditis elegans.Prediction of coiled-coil domains in related MED15 and Pum1 proteins. Coiled-coil domains in proteins were predicted using the COILS program with a window size of 28 amino acids. (A) Predicted CC domain of human MED15 and its orthologues. (B) Predicted CC domain of human Pum1 and its orthologues. Abbreviations: hs, (TIF)Click here for additional data file.Figure S8Schematic representation of a cell-based LUMIER co-immunoprecipitation assay. To investigate the interaction between the proteins ATXN1Q30 (bait) and Pum1 (prey) the fusion protein pairs (A) PA-RL-ATXN1Q30/FL-Pum1, (B) PA-RL/FL-Pum1 and (C) PA-RL-ATXN1Q30/FL were co-produced in HEK293 cells. After 48 h the cells were lysed and protein extracts were applied to IgG coated magnetic beads  for co-immunoprecipitation (co-IP) of interacting proteins. Following the co-IP the firefly luminescence signal was determined for all three samples (A–C) using a luminescence plate reader. In sample A, as a result of the interaction between PA-RL-ATXN1Q30 and FL-Pum1 a high luminescence signal was detected. In comparison, relatively low signals were obtained in control samples B and C. Using the measured values we calculated the binding ratios R-op and R-ob, which are measure for the specificity of the ATXN1Q30 and Pum1 interaction. Based on previous empirical studies with well-characterized interaction test pairs, we define an interaction as positive when the calculated R-op and R-ob ratios are >1.5.(TIF)Click here for additional data file.Figure S9Detection of firefly luciferase and Renilla luciferase tagged fusion proteins in HEK293 cell extracts. Production of Renilla and firefly luciferase-tagged fusion proteins in HEK293 cells was monitored by quantification of luminescence activity of protein extracts. Data are shown as mean ± SD for three separate experiments performed in triplicates. Abbreviations: FL - firefly luciferase; RL - Renilla luciferase; PA - protein A; RLU - relative light units.(TIF)Click here for additional data file.Figure S10E. coli and purified by affinity chromatography. Analysis by SDS-PAGE and Coomassie staining confirmed that recombinant fusion proteins with expected sizes are produced in E. coli.Purification of recombinant GST- and His-tagged fusion proteins for cell-free aggregation assays. (A) Schematic representation of a spontaneous ATXN1Q82 aggregation reaction in the presence of a modifier protein. Aggregation of ATXN1Q82 is initiated by proteolytic cleavage of the fusion protein GST-ATXN1Q82 with PreScission protease. Insoluble ATXN1Q82 aggregates form spontaneously over time and are quantified by filter retardation assay. The effects of modifiers can be assessed in a time and concentration dependent manner. (B) GST- and His-tagged fusion proteins were produced in (TIF)Click here for additional data file.Figure S11The recombinant proteins His-MED15 and His-Pum1 influence spontaneous ATXN1Q82 aggregation in a concentration-dependent manner. (A) Detection of SDS-insoluble ATXN1Q82 aggregates by filter retardation assay. GST-ATXN1Q82 (5 µM) was incubated with PreScission protease and modulator proteins  and formation of insoluble ATXN1Q82 protein aggregates was quantified after 48 h by filter assay. His-MED15 in a concentration dependent manner increased spontaneous ATXN1Q82 aggregation, while His-Pum1 has the opposite effect. SDS-insoluble ATXN1Q82 protein aggregates retained on filter membranes were detected using the anti-ATXN1 antibody SA4645. (B) Quantification of filter retardation assay data with the AIDA densitometry software. Error bars represent SD of three independent experiments.(TIF)Click here for additional data file.Figure S12NT protein (Effects of modulator proteins on aggregation of full-length YFP- ATXN1Q82. (A) Effects of mCherry-tagged proteins Pum1-MED15CC, Pum1, MED15, S100B, STUB1 and luciferase on spontaneous YFP-ATXN1Q82 aggregation in human neuroblastoma SH-EP cells. The formation of polyQ-containing ATXN1 aggregates was quantified by fluorescence imaging after 48 h. We found that the hybrid protein Pum1-MED15CC in comparison to the wild-type Pum1 and the luciferase control protein (100%) readily promotes YFP-ATXN1Q82 aggregation, confirming the results obtained with the YFP-ATXN1Q82 protein . An incr(TIF)Click here for additional data file.Figure S13Production of mCherry- and FLAG-tagged modifier proteins in mammalian cells. SH-EP neuroblastoma cells were transfected with plasmids encoding (A) mCherry- or (B) FLAG-tagged modifier proteins; protein extracts were analyzed after 48 h by SDS-PAGE and immunoblotting. Recombinant proteins with the expected sizes were detected with an anti-mCherry (A) or an anti-FLAG antibody (B). The endogenous protein actin was used as a loading control.(TIF)Click here for additional data file.Figure S14NT, ATXN1Q82NT, ATXN3Q25 or ATXN3Q73 fusion proteins and cell extracts were analyzed by SDS-PAGE and immunoblotting using the anti-ATXN1 antibody SA4645. We observed that all YFP-tagged ATXN1 and ATXN3 fusion proteins were detected with an anti-GFP antibody (A). In contrast, the SA4645 antibody detects ATXN1 but not ATXN3 fusion proteins in cell extracts (B), indicating that the antibody is specific for ATXN1. An anti-actin antibody was utilized to detect endogenous actin in cell extracts (loading control). Abbreviations: IB – immunoblot; a-GFP – anti-GFP antibody; a-ATXN1 - anti-ATXN1 antibody SA4645.The SA4645 antibody specifically recognizes YFP-tagged ATXN1 fusion proteins in crude cell extracts. COS-1 cells were transiently transfected with plasmids encoding YFP-tagged ATXN1Q30, ATXN1Q82, ATXN1Q30(TIF)Click here for additional data file.Table S1List of human genes encoding potential ATXN1 toxicity and aggregation modifiers. Genes encoding putative modifier proteins were predicted using the available literature information from modulator studies in lower model organisms. In addition, interaction partners of polyQ-containing disease proteins  were selected as potential modulator proteins.(XLS)Click here for additional data file.Table S2NT toxicity or aggregation modulators. The genes encoding YFP-ATXN1Q82NT toxicity modulators were initially identified in cDNA overexpression experiments in COS-1 cells. In further studies their effects on YFP-ATXN1Q82NT aggregation was investigated. Finally, we examined whether identified YFP-ATXN1Q82NT toxicity modulators also influence YFP-ATXN3Q73 toxicity and aggregation in COS-1 cells.Human genes encoding YFP-ATXN1Q82(XLS)Click here for additional data file.Table S3NT toxicity modifiers were investigated in cell-based siRNA knock-down experiments.Selected genes for siRNA knock-down experiments in cell-based assays. 15 genes encoding potential ATXN1Q82(XLS)Click here for additional data file.Table S4NT toxicity modifiers. ATXN1Q82NT toxicity modifiers were categorized using the available literature information retrieved from PubMed.Literature-based annotation of potential cellular functions of ATXN1Q82(XLS)Click here for additional data file.Table S5NT toxicity modifiers. Coiled-coil (CC) domains in modifier proteins were predicted using the COILS program and a window size of 28 amino acids.Predicting coiled-coil domains in ATXN1Q82(XLS)Click here for additional data file.Table S6NT and ATXN1Q82NT.Oligonucleotides for PCR amplification of cDNA fragments. PCR amplified cDNA fragments were utilized for constructing Gateway compatible entry plasmids. We generated cDNA fragments for the proteins MED15CC, MED15ΔCC, ATXN3Q25, ATXN3Q73, ATXN1Q30, ATXN1Q82, ATXN1Q30(XLS)Click here for additional data file.Table S7List of cDNA entry clones for construction of destination plasmids.(XLS)Click here for additional data file.Table S8E. coli and mammalian cells.List of destination plasmids for expression of cDNAs in (XLS)Click here for additional data file.Table S9List of oligonucleotides for qPCR experiments.(XLS)Click here for additional data file.