Background: Membrane-associated UCH-L1 has been proposed as a target for treating neurodegeneration.Results: UCH-L1 association with neuronal membranes does not involve farnesylation. C-terminal truncation causes protein misfolding and neuronal death.Conclusion: The C terminus of UCH-L1 regulates protein aggregation and stability.Significance: The CTTΔ4 mutant provides a model for studying neurotoxic UCH-L1 aggregation events. C) and a membrane-associated form (UCH-L1M). Alzheimer brains show reduced levels of soluble UCH-L1C correlating with the formation of UCH-L1-immunoreactive tau tangles, whereas UCH-L1M has been implicated in α-synuclein dysfunction. Given these reports of divergent roles, we investigated the properties of UCH-L1 membrane association. Surprisingly, our results indicate that UCH-L1 does not partition to the membrane in the cultured cell lines we tested. Furthermore, in primary cultured neurons, a proportion of UCH-L1M does partition to the membrane, but, contrary to a previous report, this does not require farnesylation. Deletion of the four C-terminal residues caused the loss of protein solubility, abrogation of substrate binding, increased cell death, and an abnormal intracellular distribution, consistent with protein dysfunction and aggregation. These data indicate that UCH-L1 is differently processed in neurons compared with clonal cell lines and that farnesylation does not account for the membrane association in neurons.Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed in neurons. A possible role for UCH-L1 in neurodegeneration has been highlighted because of its presence in Lewy bodies associated with Parkinson disease and neurofibrillary tangles observed in Alzheimer disease. UCH-L1 exists in two forms in neurons, a soluble cytoplasmic form (UCH-L1 Ubiquitination is a dynamic, posttranslational modification that is fundamentally important in controlling multiple aspects of neuronal function, including turnover, trafficking, and synaptic plasticity , 2. Indein vivo remain to be determined.Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a highly abundant neuronal deubiquitinase, a cysteine protease that cleaves small peptide adducts from the C terminus of ubiquitin. Despite the fact that UCH-L1 comprises 1–5% of total neuronal protein , the widin vitro, especially in comparison with the more widely expressed UCH-L3, with which it shares 52% sequence homology (UCH-L1 binds ubiquitin with high affinity but possesses poor hydrolase activity homology . Aside fhomology .C) and membrane (UCH-L1M) fractions. In neurons, it has been estimated that 20–50% is membrane-associated  caused the loss of ubiquitin substrate binding, increased neuronal death, altered UCH-L1 distribution within neurons, and removed soluble, cytoplasmic UCH-L1 with a corresponding increase in Triton X-100-insoluble UCH-L1. A proportion of truncated UCH-L1 was still detected in the membrane fraction, but the levels of soluble cytosolic UCH-L1 were significantly decreased. Circular dichroism analysis of WT and a C-terminal truncation mutant (CTTΔ4) revealed that the removal of the last four residues of UCH-L1 caused a minor disruption to the secondary structure but dramatically increased aggregation.These changes are in line with previous reports on the disruption to the tertiary structure and aggregation of UCH-L1 and are consistent with the distribution and soluble properties of UCH-L1 in Alzheimer brains , 15, sugPrimary hippocampal and cortical neuronal cultures were prepared from embryonic day 18 rats exactly as described previously . The N2aAll SDS-PAGE and Western blotting procedures were carried out according to standard protocols. The following primary antibodies were used: mouse anti-UCH-L1 , rabbit anti-GluA1 , rabbit anti-RhoGDI , mouse anti-H-ras , mouse anti-Rac1 , rabbit anti-HA , goat anti-Lamin B , and rabbit anti-PSD95 . All blots were developed using Li-Cor Odyssey imaging software, except for anti-Lamin B, which was developed on film using HRP-conjugated anti-goat . For Li-Cor Odyssey Western blot imaging, the IRDye secondary antibodies  used were 800CW donkey anti-mouse, 800CW donkey anti-rabbit, 680RD donkey anti-mouse, and 680RD donkey anti-rabbit, all 1:10,000. Western blot signal quantification was performed using Li-Cor Odyssey imaging software.Fractions were obtained as described previously .m sucrose, 20 mm HEPES (pH 7.4), 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm DTT, and protease inhibitors (Roche)), lysed by passing the cell suspension through a 25-gauge needle 12 times using a 1-ml syringe, and then sonicated briefly three times on low power. Lysates were centrifuged at 100,000 × g in a benchtop ultracentrifuge (Beckman) for 1 h at 4 °C. The supernatant was retained as the cytosolic fraction. The pellet was washed three times in subcellular fractionation buffer and resuspended in a standard lysis buffer containing 1% Triton and 0.1% SDS and solubilized on a rotating wheel at 4 °C for 30 min. The suspension was centrifuged at 10,000 × g in a benchtop centrifuge for 10 min at 4 °C. The supernatant was retained as the membrane fraction. The Triton X-100-insoluble fraction was prepared by resuspending the remaining pellet in lysis buffer containing 1% Triton and 0.1% SDS, rotated at 4 °C for 30 min, passed through a 25-gauge needle 12 times using a 1-ml syringe, and then sonicated three times on low power. Protein concentration was determined using a Bradford assay (Bio-Rad) to determine protein concentration compared with a set of BSA standards. Sarkosyl solubility was determined by resuspending the Triton X-100-insoluble fraction in lysis buffer containing 1% N-lauroylsarcosine sodium salt (Sigma), passed through a needle and sonicated as before, rotated at room temperature for 1 h, and ultracentrifuged at 100,000 × g at 4 °C for 45 min. 20 μl of the supernatant was loaded on the gel as the sarkosyl-soluble fraction. The remaining pellet was resuspended in lysis buffer containing 2% SDS, passed through a needle, resonicated, and then rotated on a wheel at room temperature for a further 30 min. 20 μl was loaded on the gel as the sarkosyl-insoluble fraction.Cells were scraped in detergent-free subcellular fractionation buffer  or the farnesyltransferase inhibitor FTI-276  for 48 h prior to undergoing the subcellular fractionation protocol at day The GFP-UCH-L1 human cDNA plasmid was a gift from Prof. Mike Clague (University of Liverpool). His6-TAT-HA-UCH-L1 was a gift from Dr. Ottavio Arancio. HA-UCH-L1 constructs were generated using standard cloning procedures and PCR-based mutagenesis. Sindbis virus expressing HA-UCH-L1 constructs were created using the pSinRep5 plasmid and Ambion virus production tools.m of UbVMECultured cortical neurons were infected with Sindbis virus-expressing HA-UCH-L1 constructs for 24 h prior to lysis. N2a cells were transfected with HA-UCH-L1 constructs at ∼75% confluency using Lipofectamine LTX Plus (Invitrogen) for 40 h prior to lysis. 20 μg of cell lysates was incubated with 2 μin vitro 18 dissociated hippocampal neurons infected with Sindbis virus expressing HA-UCH-L1 constructs for 24 h. Anti-UCH-L1 and anti-HA antibodies were used at 1:500. Fluorophore-conjugated secondary antibodies used were goat anti-mouse-DyLight 488 and goat anti-rabbit DyLight 649 (Jackson ImmunoResearch Laboratories) and Hoechst nuclear stain . Confocal imaging was performed using a ×63 oil immersion objective lens  on a Zeiss LSM 150 META inverted confocal microscope. Maximum projection confocal z stacks were taken using 0.4-μm steps using a 1024 × 1024 pixel resolution and a pinhole ∼1 airy unit and prepared using ImageJ software .For fixed cell confocal imaging, standard 4% PFA fixation and immunostaining protocols were used on day in vitro toxicology assay kit (Sigma). Briefly, day in vitro 18 dissociated cortical neurons cultured in 3-cm dishes were infected with Sindbis virus expressing HA-UCH-L1 constructs for 24 h. 100 μl of conditioned culture medium was taken from each dish. 30 μl of medium was transferred to each well of a 96-well plate. A lactate dehydrogenase assay mixture containing substrate, cofactor, and dye solutions was prepared according to the instructions of the manufacturer, and 60 μl of the mixture was added to each well of a 96-well plate. Following 25 min of incubation, the reaction was quenched using 10 μl of 1 N HCl, and the absorbance at 492 nm and at reference 620 nm was measured using a Tecan program on a plate reader.lactate dehydrogenase levels in conditioned neuronal culture media were assessed using an m in PBS with 1 mm Tris (2-carboxyethyl)phosphine and 0.01% SDS. CD data were collected on a Jasco J810 spectropolarimeter fitted with a Peltier temperature-controlled sample stage. Samples were placed in a 1-mm path length quartz cuvette (Starna), and the scan rate was 50 nm min−1 with a 1-nm interval, a 1-nm bandwidth, and a 1-s response time.Purified TAT-HA-UCH-L1 and TAT-HA-CTTΔ4 were analyzed by circular dichroism spectroscopy at a concentration of 5 μA). Immunoblotting for the cytosolic marker RhoGDI showed that the presence of UCH-L1 in these fractions was not due to cytosolic contamination.Subcellular fractionation of adult rat brain was used to identify the neuronal compartments that contain UCH-L1. As expected, UCH-L1 was most abundant in the cytosolic fractions, but it was also detected in several of the membrane-enriched fractions A. ImmunoB). Importantly, we also confirmed that this protocol preserves the cytosolic/membrane ratio of proteins that partition into both compartments by analyzing Rac1. Rac1 is a partially prenylated protein that is posttranslationally modified by geranylgeranylation (B). UCH-L1 was detected by Western blot in both the cytosolic and membrane fractions (B) with ∼30% UCH-L1M (C), consistent with values reported previously and, potentially, for a role of farnesylation in regulating membrane partitioning  and membrane (GluA1) protein markers B. Importnylation  leading nylation B. UCH-L1ractions B with ∼3 UCH-L1M C, consisitioning , 11.AAX , with the resulting cleavage of the final three residues  compared with cultured cortical neurons (∼32.2%) (D and E). To test whether the proportion of UCH-L1M in neurons is linked to neurite outgrowth, we induced N2a cell differentiation. 24 h of serum starvation produced neuronal-like processes characteristic of differentiation, but the proportion of UCH-L1M was unchanged (F and H). Taken together, our data strongly suggest that membrane association occurs selectively in neurons but is not dependent on differentiation.Farnesylation involves the irreversible posttranslational addition of a 15-carbon lipid tail to the cysteine at the C-terminal end of proteins containing the sequence Cresidues . The memresidues . However(∼32.2%) , D and Enchanged , F and HM to the farnesyltransferase inhibitor FTI-276. Because membrane-association of H-ras is mediated by farnesylation at the C terminus .Several posttranslational modifications that generate membrane association, such as prenylation (farnesylation and geranylgeranylation) and palmitoylation, rely on covalent attachments to cysteine residues. We therefore mutated three cysteine residues in UCH-L1 (3CS) Cys-90, Cys-152, Cys-220) because these side chains are likely accessible for modification in the cytosol. Although Cys-90 is part of the catalytic triad in the active site, Cys-152 can bind lipid products that mediate the cellular response to oxidative stress and inflammation A.B). These data suggest that mutation of any individual cysteine residue does not disrupt UCH-L1 conformation.Mutation of each of the six cysteine residues in HA-tagged UCH-L1 was tested in N2a cells to assess whether UCH-L1 could still bind covalently to a ubiquitin vinyl methyl ester suicide substrate (Boston Biochem), which forms a bond with Cys-90 in the active site. Notably, the C152S and C220S mutants, individually or combined, showed comparable substrate binding compared with the WT, whereas binding was abolished in the C90S mutant B. These C, HA-UCH-L1 bound to UbVME, and ∼25% partitioned into the membrane, consistent with ∼30% of endogenously expressed UCH-L1 (D and E). Therefore, the HA-tagged virally overexpressed UCH-L1 behaves in a similar manner as endogenous protein. Interestingly, ∼20% of the HA-UCH-L1–3CS mutant was also present in the membrane fraction, suggesting that the cysteine modification at Cys-90, Cys-152, or Cys-220 is not involved in membrane partitioning.We next infected cultured cortical neurons with WT or the triple cysteine mutant HA-UCH-L1 (3CS). As shown in Fig. 3C), although the overexpressed HA-UCH-L1-D30K mutant still partitioned into the membrane fraction (∼22%) . Therefore, together with the data from the 3CS mutant, we conclude that membrane association of UCH-L1 is not dependent on an interaction with the ubiquitin-binding region of UCH-L1.Because UCH-L1 has a high affinity for ubiquitin, we tested whether membrane association of UCH-L1 might arise from binding to a protein containing a domain resembling a ubiquitin fold. The Asp-30 site on UCH-L1 is required for ubiquitin binding . The D30Fig. 3M. Our results indicate that I93M partitions into the membrane and retains sufficient overall structure to bind UbVME to the same extent as WT UCH-L1.The I93M UCH-L1 point mutation has been linked to a familial strain of Parkinson disease . InteresA). UCH-L1 has a complex “knotted” backbone structure, and it has been predicted that even minor N- or C-terminal truncations would denature the protein (B). Furthermore, there was no significant change to the proportion of HA-UCH-L1 in the membrane fraction with either the N-terminal truncation (5–223) or the C-terminal truncation  (C).To investigate the possible roles of signaling sequences at the N and C termini in UCH-L1 membrane association, we removed the first four N-terminal amino acids (MQLK) and the final two residues (AA) A. UCH-L1 protein . Our dat protein B. Furthe, 1–221) C.B), suggesting that the protein conformation had been altered. Consistent with this, there was a significant loss of the soluble cytosolic form of UCH-L1, with the majority of the signal being detected in a 1% Triton X-100-insoluble fraction. There was also an increase in the proportion of UCH-L1M in neurons infected with the CTTΔ4 mutant compared with the WT (C and D).Although the CTTΔ2 truncation behaved the same as the WT, an HA-tagged C-terminal truncation , which deleted the final four residues (CKAA) encompassing the proposed site of farnesylation, reduced cell viability. Initially we noticed that UbVME binding was abolished B, suggesh the WT , C and DE).It has been reported that UCH-L1 is enriched in the insoluble protein aggregates, consistent with several types of neurodegenerative disease . To inveFig. 4A). In each case, the protein is approximately one-third α-helical. The most significant difference between the proteins is the marked increase in aggregation of truncated UCH-L1, as shown by the greatly increased scattering evident in the spectrum, especially at lower wavelengths.To explore the possible reasons for the changes in solubility, we analyzed the secondary structure using circular dichroism. The spectra show that the truncated protein has a shallower minimum at 222 nm, indicating a slightly reduced α-helical content compared with the WT A. In eacA). Furthermore, overexpression of this mutant markedly increased neuronal cell death, as measured by lactate dehydrogenase release (B).Confocal microscopy of hippocampal neurons infected with the different UCH-L1 constructs using Sindbis virus revealed significant differences in the distributions of CTTΔ4 compared with the WT. CTTΔ4 displayed a non-uniform, mostly perinuclear staining and was largely absent from the processes A. Furthe release B.M). A previous report proposed that farnesylation mediates membrane association in cell lines  with which UCH-L1 associates has remained unclear because the very high levels of expression of native UCH-L1 in neurons, combined with its relatively uniform distribution, make it difficult to resolve using imaging techniques such as confocal microscopy. Also, unlike the previous report in clonal cell lines  caused a dramatic increase in neurotoxicity, whereas deletion of the final two residues  had no marked effect . The I93M mutation is associated with Parkinson disease and has been reported to decrease UCH-L1 solubility and hydrolytic activity (D).A common theme that emerges from both point mutation and oxidative modification studies of the misfolding and/or aggregation of UCH-L1 is disruption to the central hydrophobic core , 31. AltFig. 5activity , 31. Theactivity . Howeveractivity . Overallactivity D.B). Analogous to this disruption of the packed core structure by prostaglandins, our results indicate that disruption of the extreme C terminus may also have a similar effect and provide a potential mechanism for how UCH-L1 structure and stability may be regulated by other forms of oxidative stress.Cyclopentenone prostaglandins such as 15d-PGJ2 are produced during an immune response or under oxidative stress. 15d-PGJ2 specifically modifies UCH-L1 at C152 site, leading to a loss of protein stability B. Analog2ETL, Ref. Crystal structures of UCH-L1 suggest that Lys-221 might be integral to the tertiary structure by forming intramolecular bonds to hold the three-dimensional shape of the protein (PDB code We therefore propose that exposure of the hydrophobic core causes a toxic gain of function in which misfolded UCH-L1 aberrantly binds to hydrophobic intracellular membranes, and this leads to cell death. The CTTΔ4 mutant we report here represents an extreme form of unfolded UCH-L1. Importantly, we attribute this to a toxic gain of function rather than a loss of hydrolytic activity because C90S and D30K do not have the same effect. Taken together, our findings suggest that modifications or mutations that alter the folding and/or stability of UCH-L1 are neurotoxic. The CTTΔ4 truncation provides a useful new tool for studying how UCH-L1 misfolding and dysfunction lead to protein instability and cell death, which pathways are involved, and what measures can be taken to reduce or prevent toxicity.In conclusion, our results demonstrate that membrane association of UCH-L1 is differentially regulated in different cell types. We investigated a series of possible mechanisms for the membrane association, and our data indicate that, contrary to a previous report, farnesylation and/or other cysteine posttranslational modifications are not responsible for membrane partitioning. Therefore, further work is required to define how UCH-L1 is recruited and retained at membranes selectively in neurons and to elucidate the functional consequences of this membrane association.


Artemia has evolved a special reproductive mode to release encysted embryos in which cell division can be maintained in a dormancy state for a long period.In eukaryotes, tRNA trafficking between the nucleus and cytoplasm is a complex process connected with cell cycle regulation. Such trafficking is therefore of fundamental importance in cell biology, and disruption of this process has grave consequences for cell viability and survival. To cope with harsh habitats, Artemia as a peculiar model of the cell cycle, an La-related protein from Artemia, named Ar-Larp, was found to bind to tRNA and accumulate in the nucleus, leading to cell cycle arrest and controlling the onset of diapause formation in Artemia. Furthermore, exogenous gene expression of Ar-Larp could induce cell cycle arrest in cancer cells and suppress tumor growth in a xenograft mouse model, similar to the results obtained in diapause embryos of Artemia. Our study of tRNA trafficking indicated that Ar-Larp controls cell cycle arrest by binding to tRNAs and influencing their retrograde movement from the cytoplasm to the nucleus, which is connected to pathways involved in cell cycle checkpoints.Using Artemia offer new insights into the mechanism underlying cell cycle arrest regulation, as well as providing a potentially novel approach to study tRNA retrograde movement from the cytoplasm to the nucleus.These findings in The online version of this article (doi:10.1186/s12915-016-0239-4) contains supplementary material, which is available to authorized users. INK4a, which binds to CDK4 and arrests the cell cycle in G1 phase, and p14Arf in humans, which prevents p53 degradation [Regulation of the cell cycle involves processes crucial to survival, including the detection and repair of genetic damage and the prevention of uncontrolled cell division. This subject has attracted much attention and has enormous practical implications for combating cancer. The cell cycle has several well-controlled phases. Progression into each phase is tightly regulated by checkpoints and inhibitors , 2. The Saccharomyces cerevisiae, un-spliced tRNA rapidly accumulates in the nuclei, and cell cycle progression is delayed at the G1 to S phase transition [In eukaryotes, tRNA trafficking between the nucleus and the cytoplasm is a complex process that often responds to environmental stress, thereby connecting transcription in the nucleus to translation in the cytoplasm . This transition , 8. In cansition –12. Retransition . The regansition  and is dansition .La proteins are confined to the nucleus and the complete evolutionary in eukaryotic cells. Structural analysis of these proteins resulted in their classification into five families, namely the genuine La homologs and four Larp families: Larp1, 4, 6, and 7 , 17. La Artemia model studied here is found in severely hypersaline environments. Under certain conditions, mature females produce and release encysted gastrula embryos  that enter diapause, a state of obligate dormancy. Different environmental cues lead to uninterrupted (direct) embryonic development, resulting in the release of swimming nauplius larvae [Artemia model depicts adaptation as a complex response to critical life conditions, integrating and refining past and present experiences at all levels of organization [The s larvae . A featus larvae , 21. Dias larvae , 21. Rems larvae , and eves larvae . The Artnization .Artemia diapause was used as a cell cycle arrest model. In this study, an RNA-binding and La-related protein, named Ar-Larp, was found to accumulate in the nucleus in response to cell cycle arrest, which resulted in the formation of Artemia diapause by binding to tRNAs. The mechanisms underlying the regulation of cell cycle arrest by Ar-Larp were elucidated in cancer cells using exogenous Ar-Larp gene transfection and expression. Cell cycle arrest induced by tRNA retrograde movement from the cytoplasm to the nucleus was then demonstrated in cancer cells. Our results indicated that tRNA trafficking regulates the mitogenesis and proliferation of cells through cell cycle checkpoints, a process that is mediated by multiple signaling pathways including histone H3 acetylated at lysine 56 (H3K56ac), extracellular signal-regulated kinase (ERK), and Akt. Ar-Larp is thus an upstream signal of tRNA trafficking that regulates cell cycle progression in response to environmental stresses.To elucidate the molecular mechanism underlying cell cycle arrest and its link to the regulation of tRNA nucleocytoplasmic trafficking, Artemia possesses two independent reproductive pathways that allow adaptation to widely fluctuating environments. Under unfavorable conditions, mature females produce and release encysted embryos that enter diapause, a state of obligate dormancy  protein , designed from the Ar-Larp cDNA sequence, was injected into the body cavity of adult oviparous Artemia. One day after the formation of diapause embryos (about 2 weeks post-injection), Ar-Larp mRNA levels decreased in proportion to increasing doses of the dsRNA  was used to knockdown RNA Fig. . After ilii Fig. . In genelii Fig. . These rArtemia diapause formation, previous studies have focused on molecules associated with extreme environmental tolerance. For example, the small heat shock/α-crystallin protein p26, which acts as a molecular chaperone, is expressed in diapause cysts and is transported into nuclei during periods of environmental stress [Ar-Larp gene prevented the formation of a complete diapause state and inhibited expression of p26 and artemin  and tRNAs using an electrophoretic mobility shift assay (EMSA). The results showed that GST-Ar-Larp could form a complex with tRNA in vitro, and the complex was identified as a super-shifted band when an anti-Ar-Larp antibody was incubated together with Ar-Larp and tRNAs Fig. . To clarArtemia oocytes are unusual cells, in that large quantities of proteins and RNAs are stored in preparation for early development. Thus, results obtained in Artemia oocytes may differ from other kinds of cells.Both genetic and biochemical studies have revealed that a major role of La is to protect the 3’ end of nascent small RNAs from exonuclease digestion , 31. AltArtemia. Ar-Larp-tRNA complexes were identified in diapause embryos of Artemia and could be detected using several tRNA probes . Based on these results, we suggest that Ar-Larp controls cell cycle arrest in diapause by interacting with tRNAs in the nucleus.Next, co-immunoprecipitation (co-IP) was performed to investigate whether this interaction occurred in bes Fig. . HoweverArtemia, green fluorescent protein (GFP)-fused Ar-Larp was overexpressed in the human cancer cell lines HeLa  was constructed and injected into HeLa and HT1080 human tumor xenograft model mice. Western blotting revealed the presence of Ar-Larp in tumor specimens, indicating that the protein was successfully expressed from the adenovirus construct in vivo Fig. . ImportaAr-Larp was transfected into HeLa cells and tRNA trafficking was analyzed. Fluorescence in situ hybridization (FISH) using tRNA-specific probes was used to examine the distribution of tRNAs. The results indicated that tRNAs accumulated significantly in the nuclei of Ar-Larp-transfected HeLa cells compared to the controls  was used to block de novo tRNA synthesis. In complete medium, tRNA could not be detected in the nuclei of control cells treated with ActD; however, tRNA accumulation in the nuclei was observed in Ar-Larp-overexpressing cells treated with ActD Fig. . During S. cerevisiae cells in response to aa deprivation [Although the regulation of post-transcriptional gene expression by tRNA nuclear accumulation remains an unproven possibility, evidence indicates that retrograde tRNA nuclear accumulation occurs in response to environmental stress. Previous studies reported that tRNA moves retrogradely from the cytoplasm to the nucleus in rat hepatoma and rivation , 33, 34.Artemia contains a La motif and two RRMs that are also found in La protein and LARP7. However, we found that Ar-Larp shows only 18 % and 17 % sequence identities to human La protein and LARP7, respectively  were raised in 8 % (w/v) artificial seawater (Blue Starfish) at 25 °C on a 4L:20D cycle, while the directly developing Artemia were reared in 4 % (w/v) artificial seawater on a 16L:8D cycle. Chlorella powder  was supplied as brine shrimp food. BALB/c nude mice aged 6–8 weeks  were used as xenograft recipients for cancer cells. Animal experiments were approved and performed in accordance with the institutional guidelines for animal care of the animal ethics committee of Zhejiang University.Diapause-destined 2 in DMEM, RPMI-1640, and EMEM , respectively, supplemented with 10 % fetal bovine serum . Cell transfections were performed with Lipofectamine 2000  as recommended by the manufacturer.HeLa, HT1080, and MKN45 cells  were cultured at 37 °C in 5 % COEscherichia coli BL21 (DE3) and purified using Ni-NTA Agarose . The anti-Ar-Larp antibody was raised in rabbits . For nuclear and cytoplasmic fractions, the extracts were prepared using NPE Nuclear and CPE Cytoplasmic Extraction Reagents  following the manufacturer’s instructions. Proteins were extracted from each tissue using protein loading buffer  sodium dodecyl sulfate (SDS), 0.2 % (w/v) bromophenol blue, and 20 % (v/v) glycerol prepared in 0.1 mM Tris-HCl, pH 6.8). Fifty micrograms of protein of each sample were separated on 12.5 % SDS-PAGE gels and transferred to PVDF membranes . The membranes were incubated with antibodies overnight at 4 °C and detected using the BM Chemiluminescence Western Blotting Kit  following the manufacturer’s instructions.Ar-Larp protein with His-tag was expressed in t-test. The differences were considered statistically significant at P <0.01.Fluorescence-activated cell sorting was performed as previously described . Data anIn vivo BrdU labeling was performed according to the manufacturer’s instructions . Cells were incubated with 12 mM BrdU (Sigma) for an additional 24 h after transfection. The incorporation of BrdU into the genome was detected with a mouse anti-BrdU primary antibody (Sigma) followed by a secondary goat anti-mouse antibody conjugated to rhodamine (HuaAn). Wide-field fluorescence microscopy was used to obtain images.t-test.Total RNA was isolated using TRIzol reagent (Invitrogen). cDNA was synthesized from 2 μg of total RNA using a SuperScript™/First-Strand cDNA Synthesis kit (Invitrogen) with Oligo(dT) primers. cDNA was diluted 10-fold and combined with SYBR Green Supermix  for quantitative PCR. The relative abundance of mRNA was normalized to that of α-tubulin. The results are reported as the mean ± SD of three independent experiments. Comparisons between groups for statistical significance were performed with a two-tailed paired Student’s Artemia using an UltraMicroPump II equipped with a Micro4™ MicroSyringe Pump . Similarly, GFP dsRNA was injected as the negative control.Recombinant plasmids expressing Ar-Larp dsRNAs were constructed and the dsRNAs were induced, expressed, and purified as described previously . Ar-LarpS rRNA, 5S RNA, and mRNAs) were added to the EMSA system.For the EMSA, yeast tRNAs  were first labeled with the digoxigenin (DIG) Oligonucleotide 3'-End Labeling Kit  and then incubated with various concentrations of a GST-Ar-Larp fusion protein (0–1 μg) or GST alone (1 μg) in 20 μL of RNA-binding buffer (Tris (pH7.5) 0.1 M, KCl 0.5 M, dithiothreitol 10 mM, with diisodecyl glutarate) at room temperature for 30 min. The reaction mixtures were electrophoresed on 12.5 % non-denaturing polyacrylamide gels in Tris/Borate/EDTA buffer, and detected using the Northern Blotting Kit  following the manufacturer’s instructions. For the super-shift assay, an anti-Ar-Larp antibody (0.5–1 μg) was incubated simultaneously in the EMSA system. For competition EMSA experiments, several excess competitors  and the RiboLock™ Ribonuclease Inhibitor , and were incubated with an anti-Ar-Larp antibody (HuaAn) at 4 °C overnight, followed by the addition of Protein A Dynabeads (Invitrogen) for 3 h at 4 °C. The beads were washed with extraction buffer twice, and tRNAs were purified using TRIzol reagent (Invitrogen). RNA was loaded onto 8 % urea denaturing polyacrylamide gels prior to hybridization with tRNA oligonucleotide probes at 39 °C.A recombinant adenovirus expressing Ar-Larp (Ad-Ar-Larp) was constructed and purified using the Adenovirus Dual Expression Kit (TaKaRa) following the manufacturer’s instructions. Tumor-bearing BALB/c nude mice were injected with Ad-Ar-Larp or a control adenovirus (Ad-control) twice a week. Tumor volumes were determined by measuring tumors in two dimensions as described previously . Four wetRNA FISH was performed as described previously . To blocSupporting information and additional methods can be found in Additional files


Prostate cancer cell proliferation and hypoxia-induced angiogenesis are also regulated by the SUMO pathway, through an AR-independent mechanism. Thus, an important role has been revealed for members of the SUMO pathway in prostate cancer (PCa) development and progression, offering new therapeutic targets.SUMO  conjugation is a reversible three-step process of protein post-translational modifications mediating protein-protein interactions, subcellular compartmentalization and regulation of transcriptional events. Among divergent transcription factors regulated by SUMOylation and deSUMOylation, the androgen receptor (AR) is of exceptional significance, given its established role in prostate carcinogenesis. The enzymes of the SUMO pathway can have diverse effects on AR transcriptional activity, either via direct modification of the AR or through modification of AR co-regulators. Accumulating  Prostate cancer (PCa) is the most common cancer and the second leading cause of death from cancer in males in most western countries. The androgen receptor (AR) is one of the most important factors in PCa development . The traIn vitro evidence has indicated that Ubc9 is sufficient for binding to the SUMO acceptor site and efficiently transferring SUMO to selected targets [in vivo. Three classes of proteins have been identified to have SUMO E3 ligase activity: the protein inhibitor of activated STAT (PIAS) family proteins [in vivo and in vitro [SUMOylation represents one such important post-translational modification system that regulates the activity of many transcriptional regulators . The con targets . Howeverproteins ,11, the proteins , and Ranproteins . The PIAin vitro ,15.SUMOylation is readily reversible in the cell because the isopeptide bond created between the C-terminal glycine in SUMO and the epsilon amino group in the acceptor lysine can be cleaved by SUMO proteases, which are also termed SENPs (SENtrin specific Proteases) or deSUMOylases. There are six SENP enzymes in mammals, each containing a highly conserved 200 amino acid catalytic domain that mediates deSUMOylation . The amiSUMOylation and deSUMOylation processes are responsible for transcriptional regulation of various initiated signals, including androgen-mediated transcription. However, there is an established correlation between enhanced androgen-dependence and prostate carcinogenesis and the most striking proof for this is the use of expression of the AR-regulated prostate-specific antigen (PSA) gene as a biologic marker for the diagnosis and treatment of PCa ,19,20. TThe AR can be modified by SUMOylation, preferentially by SUMO-1. Two major SUMOylation sites (K386 and K520) have been identified within the AR . The bioin vitro, probably suggesting that Ubc9 stimulation of AR transcriptional activity may be independent of its ability to catalyze SUMO-1 conjugation [Besides SUMOylation, AR is also subjected to phosphorylation and acetylation. Ubc9, the SUMO E2 enzyme, binds the AR within the hinge region  that incjugation ,26.AR activity is also regulated by the PIAS family proteins . The PIAin vivo. Studies using SUMOylation-deficient AR mutants suggested that the augmentation of AR activity by hZimp10 is dependent on the AR SUMOylation. A link was revealed between hZimp10-mediated enhancement of AR SUMOylation and modulation of AR-mediated transcription, although it remains uncertain whether hZimp10 acts directly as an E3 ligase [A novel PIAS-like protein, named hZimp10, was identified as an additional AR co-regulator. hZimp10 co-localized with AR and SUMO-1 at replication foci throughout the S phase, and it was capable of enhancing SUMOylation of AR 3 ligase ,33.The SUMOylation acceptor sites of the AR have also been mapped to a previously identified inhibitory motif called the synergy control (SC) motif . The SC But what is the molecular mechanism through which SUMOylation regulates AR transcriptional potential? It was shown that a conserved motif localized at the hinge region of the AR is responsible for acetylation and that the extent of AR SUMOylation is independent of its acetylation status , excludiN-terminal kinase in Fas-mediated apoptosis [in vitro and in vivo, combined with evidence of suppression of AR transcriptional activity by wild type, but not mutant SUMO, strongly suggest that AR SUMOylation is involved in regulating Daxx-dependent transcriptional repression [A mechanistic insight underlying SUMO-dependent transcriptional repression of the AR was provided by the implication of Daxx, initially identified as a cytoplasmic signaling molecule linking Fas receptor to Jun poptosis , in thispression . Accordipression .The most recent data on SUMO-mediated repression of AR transcriptional activity involves histone deacetylase 4 (HDAC4) binding. Intriguingly, this was found to depend on SUMOylation, rather than deacetylation, of the AR. HDAC4 increases the level of AR SUMOylation in both whole-cell and cell-free assay systems, raising the possibility that the deacetylase may act as an E3 ligase for AR SUMOylation. Knock-down of HDAC4 increases the activity of endogenous AR and androgen induction of PSA expression and PCa cell growth, which is associated with decreased SUMOylation of the receptor. Overall, HDAC4 has emerged as a new positive regulator of AR-mediated transcription, revealing a deacetylase-independent, SUMOylation-dependent mechanism of HDAC action in PCa cells .Further enhancing the diversity of SUMOylation-induced effects on AR signaling, and being at odds with the previously observed negative effect of SUMO-1 conjugation on AR-initiated transcription, SUMO-3 may have a negative or strongly positive effect on AR, depending on the type of PCa cells. In primary prostate epithelial cells, PrEC, and the PCa cells, PC-3, SUMO-3 has a weak negative effect on AR transcriptional activity. In contrast, SUMO-3 and its close relative SUMO-2 strongly enhance transactivation by endogenous AR in LNCaP cells. This positive effect is observed in both androgen-dependent and androgen-independent LNCaP cells. Mutational analysis of AR and SUMO-3 demonstrated that the SUMO-3-mediated transcriptional activation does not depend on either the previously-identified SUMOylation sites of AR or the covalent conjugation of SUMO-3 to target proteins . These rIn addition, strong clinical data regarding immunohistochemical expression of SUMO pathway member proteins in human specimens further support a critical role for protein SUMOylation in PCa development and progression. Endogenous basal PIAS3 expression was reported to localize in the nucleus in a majority of epithelial and endothelial cells. Increased PIAS3 expression was observed in 100/103 samples examined in a variety of human cancers including prostate, lung, breast, colorectal, and brain tumors. Differential PIAS3 expression and the specific patterns might therefore be useful as a molecular tumor marker .Likewise, it was found that in primary PCa, Ubc9 expression is increased compared with normal tissue, whereas in metastatic prostate tissues, it is decreased in comparison with their corresponding normal and primary adenocarcinoma tissues. Ubc9 expression correlates negatively with tumor Gleason score. The authors conclude that expression of Ubc9 is directly associated with progression of PCa, since it was high in prostatic intraepithelial neoplasia (PIN) cells and even higher in primary adenocarcinomas . To explin vitro. Both SENP1 and -2 are nuclear and found at sites proximal to AR. Their expression promotes AR-dependent transcription, but in a promoter-selective fashion. SENP1 and -2 stimulated the activity of holo-AR on compound ARE-containing promoters. The effects of SENP1 and -2 on AR-dependent transcription were dependent on catalytic activity and required intact SUMO acceptor sites in AR, indicating that their coactivating effects are mainly due to their direct isopeptidase activity on holo-AR. In PCa cells, ectopic expression of SENP1, but not that of SENP2, increased the transcription activity of endogenous AR. Silencing of SENP1 attenuated the expression of several AR target genes and blunted androgen-stimulated growth of LNCaP cells. These results indicate that SENP1 reverses AR SUMOylation and helps fine-tune the cellular responses to androgens in a target promoter-selective manner [AR SUMOylation is a dynamic process and is reversed by SENP1, which promotes AR-dependent transcription in PCa cells. Nucleocytoplasmic shuttling is an important parameter that should be always considered in SUMOylation and deSUMOylation events related to AR. Nuclear import of AR is not sufficient for SUMOylation, because constitutively nuclear apo-ARs or antagonist-bound ARs are only very weakly modified by SUMO-1 in comparison with agonist-bound ARs. Of the SUMO-specific proteases (SENP)-1, -2, -3, -5, and -6, only SENP1 and SENP2 are efficient in cleaving AR-SUMO-1 conjugates in intact cells and e manner . Intrigue manner ,42,43.SENP1 levels were enhanced in cells treated with IL-6, which is a positive regulator of the activation of AR-dependent transcription through the activation of the MAPK and JAK/STAT pathways ,45. The in vitro findings translate to an altered clinical phenotype, depending on the levels of SENP1 expression, as evidenced by induction of PIN-like structure formation in SENP1 transgenic mice that were older than 4 months [These 4 months . Elevate4 months . SENP1 m4 months .Androgen treatment of PCa cells stimulates AR SUMOylation within 15 minutes and reaches a maximum level by 1 hour . The simInvestigation of the protein expression profile of mouse prostate by the use of subcellular fractionation, 1-DE  protein separation and mass spectrometry, also demonstrated that both free SUMO-2/3 and SUMO-1 are particularly abundant in the prostate and their levels are subject to tight control by the androgen 5R-dihydrotestosterone (DHT) .DeSUMOylation is also subject to tight control by androgens, as exposure of LNCaP cells to androgen treatment enhances SENP1 transcription. This androgen-mediated augmentation of SENP1 is an AR-dependent event, as reflected by the absence of the effect both when the androgen receptor antagonist bicalutamide is present, and in AR-negative PCa PC-3 cells. A specific ARE on the promoter of the SENP1 gene is required for SENP1 induction by androgens . Thus, eIt is intriguing to speculate that androgen ablation therapy is initially effective in treating PCa due to its ability to decrease SENP1 expression. It could therefore be hypothesized that therapeutic agents designed to selectively lower SENP1 levels might be more effective than androgen ablation therapy in the treatment of advanced PCa. Like androgen ablation, reduction of elevated SENP1 levels lowers AR activity ,50 and PEmerging data implicate SUMOylation in regulating AR activity by modulating co-regulator activity. Two such factors include p68 (DDX5) and p72 (DDX17), which are members of the DEAD-box RNA helicase family that can unwind double stranded RNA and contribute to the remodeling of ribonucleoprotein complexes. These activities of p68/p72 are required for efficient RNA splicing and microRNA processing. p68/p72 perform functions that are independent of their helicase activity. This is especially common in their role as transcriptional coactivators, where p68/p72 regulate various transcription factors, including AR . One conin vitro, suggesting the notion that SUMOylation per se is not involved for binding of Alien to AR [Alien thyroid hormone receptor-interacting protein (TRIP15)/subunit 2 of the signalosome complex (CSN2) binds to the amino-terminus of AR with the receptor SUMOylation sites being involved. Both the AR N terminus with the first 328 aa and the SUMOylation sites are involved in the regulation of the Alien-AR interaction, as deletion of the inhibitory domain or mutating the SUMOylation sites of AR leads to an increase of AR-mediated transactivation. Thus, SUMOylation might have a possible role for binding to Alien, although Alien binds non-SUMOylated AR at least en to AR . Alien hen to AR .A multi-C2H2-type zinc finger protein (ZNF), ZNF451, was found to interact with both the SUMO E2 conjugase Ubc9 and SUMO-1, SUMO-2 isoforms. Further, ZNF451 interacts in a SUMO-1-enhanced fashion with AR. Although it lacks an intrinsic transcription activation function, ablation of endogenous ZNF451 in LNCaP PC cells significantly decreases expression of several AR target genes. Thus, ZNF451 acts as a transcriptional co-regulator. Both ZNF451 and AR are expressed in PCa cells. SUMOylation status of ZNF451 determines its subnuclear localization, suggesting that movable ZNF451 may influence transcription by modulating SUMO-directed trafficking of co-activator or co-repressor proteins between nucleoplasm and nuclear bodies (NBs). It is of note that AR itself evades NBs, and therefore, a “courier,” such as ZNF451, is likely to be involved in the release of co-activators from nuclear storage sites. Furthermore, ZNF451 may also tether transcriptional co-repressors, such as HDAC4, and thereby relieve repression: that is, de-repress AR activity. These data characterize ZNF451 as a novel SUMO-associated co-regulator protein that regulates androgen signaling .Pontin, a component of chromatin-remodeling complexes, is also SUMO-modified, and SUMOylation of pontin is an active control mechanism for the transcriptional regulation of pontin on AR target genes in PCa cells. Biochemical purification of pontin-containing complexes revealed the presence of the Ubc9 SUMO-conjugating enzyme that underlies its function as an activator. Intriguingly, DHT treatment significantly increased the SUMOylation of pontin, and SUMOylated pontin showed further activation of transcription of a subset of nuclear receptor-dependent target genes and led to an increase in proliferation and growth of PCa cells. These data clearly define a functional model and provide another link between SUMO modification and PCa progression. It would therefore be tempting to explore the possibility that malignant progression of PCa cells might be dependent on SUMOylation of pontin .To summarize, multiple levels of regulation of AR-dependent transcription by the SUMO pathway have been characterized in recent years, involving either direct interaction of SUMO pathway member proteins with AR or indirect control of AR-associated co-regulators . in vivo. Substitution of arginine for lysine at one putative site of SUMOylation, lysine551, blocked SUMOylation of all Sp3 isoforms in vivo and led to a marginal increase in Sp3-mediated transactivation in insect and mammalian cells. In contrast, introduction of this amino acid substitution within M1 converted it into a potent transcriptional activator. Therefore, Sp3 isoforms are SUMOylated in vivo and this post-translational modification plays an important role in the regulation of Sp3-mediated transcription. Although SUMOylation appears to only modestly reduce trans-activation by Sp3, SUMOylation of M1, and presumably M2, is required for a mechanism of transcriptional repression that is insensitive to HDAC inhibition (by trichostatin A). Regardless of the precise mechanism by which SUMOylation converts M1 and M2 into transcriptional repressors, it is evident that Sp3 isoforms are differentially regulated by SUMOylation, and the abundance of SUMOylated M1/M2 proteins determines overall levels of Sp3-mediated transcription [SUMOylation regulates the activity of prostate carcinogenesis through a variety of mechanisms, some of which are unrelated to AR signaling. For example, the Sp family of transcription factors, which contains three proteins, Sp3, M1 and M2, with differing capacities to stimulate or repress transcription. This was demonstrated in a series of experiments monitoring the activity of the natural promoter of PSA in its natural cellular milieu  in conjunction with expression vectors encoding wild-type and point-mutated Sp proteins. A yeast two-hybrid screen to identify Sp3-binding proteins resulted in the identification of Ubc9 as an M2-binding protein, and sequence analyses identified consensus SUMOylation motifs within several Sp family members. Western blots probed with anti-Sp3 detected a high molecular weight Sp3 isoform that is stabilized by a SUMO-1 hydrolase inhibitor, and this protein is also bound by anti-SUMO-1 antiserum. Transient transfection assays with epitope-tagged-SUMO-1 and GFP-SUMO-1 fusion proteins confirmed that Sp3, M1 and M2 proteins are SUMOylated cription .Provided SENP1 can strongly increase AR transcriptional activity, an effect of SENP1 on proliferation was demonstrated in androgen-dependent PCa, as endogenous SENP1 silencing by SENP1 siRNA in LNCaP cells restricted cell growth. However, similar results were observed in PC-3, an androgen-independent PCa cell line, suggesting that SENP1 might play a role in regulating cell proliferation through an AR-independent pathway. Also, the number of SENP1-silenced PC-3 cells in the G1 phase was significantly increased but was decreased in the S and G2/M phases, suggesting that SENP1 may promote G1–S phase transition in PC-3 cells. This slowing of the proliferation of SENP1-silenced PC-3 cells may thus be due to an alteration in G1–S phase progression. Silencing SENP1 expression decreased the expression of cyclin D1, but not cyclin E, in PC-3 cells. Conversely, stably transfected SENP1 in LNCaP cells enhanced cyclin D1 expression and cell proliferation. Interestingly, cell proliferation is dependent on cyclin D1 expression induced by SENP1. Thus, the regulation of cyclin D1 expression by SENP1 is another means through which PCa cell growth is regulated. The induction of cyclin D1 expression by SENP1 depended on its catalytic activity, as the mutation of SENP1 catalytic domain disrupted its activity on cyclin D1 transcription and cell proliferation [et al., 2007 reported that when HIF1α is conjugated to SUMO, its transcriptional activity is decreased and that this is not mediated by a change in the protein's half-life [Hypoxia-inducible factor (HIF) 1α stabilization, enhanced vascular endothelial growth factor (VEGF) production, and angiogenesis is regulated by SENP1 during prostate carcinogenesis. In the absence of SENP1, HIF1α is actively SUMOylated and subsequently degraded under hypoxic conditions . SENP1 aalf-life . Most imalf-life .Assessment of tissue from human PCa patients indicates elevated mRNA levels of both SENP1 and the SUMO-2/3 deconjugating enzyme, SENP3. SENP3 regulates the transcriptional activity of HIF1α via deSUMOylation of the coregulatory protein p300. Through this mechanism, overexpression of SENP3 facilitates the expression of HIF1α-regulated VEGF, which is critical for vascular development. Induction of SENP3 can be mediated via reactive oxygen species (ROS), as the latter can inhibit the ubiquitin-proteosomal mediated degradation of SENP3 to increase SENP3 protein levels ,63. HencProgression of PCa to androgen-independent growth involves many oncogenic signaling pathways, some of which are regulated by SUMOylation, as described above. Furthermore, since aberrant AR signaling (regulation of which is intimately associated with the SUMO pathway) is an important driving force of hormone resistance in PCa, the impact of the SUMO pathway may play a role in the development of androgen-refractory PCa. Further data from testing of androgen-refractory PCa cells and tissues are needed to confirm this hypothesis, however.Inevitably, many questions remain unanswered with respect to the various roles played by SUMO pathway members in PCa development and progression. For instance, does the expression of one or multiple SUMOs and SENPs fluctuate with the onset of a given carcinoma, or do specific combinations of SUMOs and SENPs alterations, regarding their function and/or localization, drive the initiation and progression of cancer? It is also difficult to tell yet whether changes in the level of SUMOylation of given transcriptional regulators persist throughout the different stages of PCa. It is possible that both SUMO conjugation and deconjugation are critical for PCa evolution. For example, one arm may be favored at an early stage to initiate tumor growth, while the other arm may be favored at more advanced stages for cancer metastasis .The potential use of elevated SENP1 or/and SENP3 levels in the prostate gland as prognostic markers could possibly identify individuals with an increased risk of developing PCa. In addition, assessment of their levels in PCa samples might serve as diagnostic markers for enhanced angiogenesis and, consequently, more aggressive carcinomas. Additional studies using tissue arrays to validate the use of either SENP1 or SENP3 as biomarkers for PCa are warranted.in vitro, with IC50 values as low as 13.0 μM detected [in vivo testing as well as in the setting of a dedicated clinical trial might lead to an effective therapeutic approach to restoring balance to the aberrant SUMO pathway of PCa patients.Attempts to target the deSUMOylation process by developing specific inhibitors for the family of SENPs are being made, although no pharmacologic agent is currently available. The first report of a successful design of SENP1 inhibitors came only recently, and involves a series of SENP1 inhibitors based on a benzodiazepine scaffold which showed inhibitory activity as high as IC50 = 9.2 μM. The compounds with the best SENP1 inhibitory activity were tested against PCa cells (PC-3) to evaluate their ability to inhibit cancer cell growth detected . Hopeful


Electronic Bingo games have recently appeared in Ontario Charitable Gaming Centres. Here we summarize the characteristics of this novel form of electronic gambling, and give a detailed characterization of one game. We contend that these games have structural characteristics that make them similar to modern Electronic Gaming Machines (EGMs) that feature multiline slots games. These features include a fast and continuous gaming experience, with player adjustable win size and reinforcement rate, a high frequency of losses disguised as wins, and highly salient near misses. Some of these games also have bonus rounds and provide players with a list of recent wins. We conclude that provincial and state gaming authorities should be aware that the placement of Bingo EGMs in existing Bingo facilities may increase problem gambling among an already well-established community of Bingo enthusiasts. Charities in Canada often raise funds using Bingo events, raffles and instant win ticket lotteries. Our home province of Ontario has experienced a dramatic drop in the popularity of its charitable gaming sector in recent years. In 2002 there were approximately 230 bingo halls helping to support nearly 6000 charities, but as of 2012 this had dropped to just 61 halls supporting 2800 charities . We played these five games for approximately 40 h.Lucky Clover Bingo: A bingo game with a traditional 5 × 5-square card with the middle square “free.” Twenty-four numbers are always called. The player wins something if the result on the card matches one or more of 53 winning patterns. The player can play from one to four cards at a time. The game is very fast, as all 24 called numbers are played automatically by the computer on all of the cards in approximately 2 s, after which any payout is added to the player’s balance.Old Glory Bingo: A bingo game that has a screen layout that is very similar to Lucky Clover but with a different set of winning patterns, a smaller maximum prize, and it is slower.The Aztec Game: A game with a 5 × 5 grid of symbols. The game has 12 lines  upon which the player can wager. The player wins if five identical items are lined up on a played line.Treasure Island: An electronic version of the paper Bingo-themed scratch ticket games that are sold in Ontario convenience stores and lottery kiosks.Berri Fruiti: An electronic version of the paper slots-themed break open tickets that are sold in Ontario convenience stories and lottery kiosks. Break open tickets are also commonly known as “Nevada tickets” or “pull tabs.”In this section we briefly describe some of the POD games we have observed and played in Ontario Charitable Gaming Centres. There were five POD games in the Charitable Gaming Centres that we visited most frequently, created by eQube Gaming .Using information freely distributed in a printed brochure about the POD games that we obtained at a Charitable Gaming Centre, plus some information given in response to our request from OLG, we collated some key parameters of these games and summarized them in Table www.multimediagames.com). We played these games for approximately 10 h. These games are called Fill’er Up, High Striker Bingo, Bingo Latte, High Sticks Bingo, Presto Bingo, and Tropical Treasures Bingo. Some notable differences are that some of these games have much higher jackpots, with Tropical Treasures Bingo having the highest jackpot at $115,000.00. These POD Bingo games also allow the player to play up to nine Bingo cards at once, as compared to Lucky Clover and Old Glory which only allow the player to play four cards. Some of these games also have bonus rounds.In some Charitable Gaming Centres we observed and played other games that were similar to the previous games, and were created by Multimedia Games . In total there are 53 different ways the 15 patterns can be arranged, and thus there are 53 possible ways of winning. The maximum prize per card is $10,000, which is achieved when the player has wagered $1.00 on a card and has covered all 12 squares of the highest paying “Clover” pattern.There are 15 winning patterns, named in the paytable on the right-hand side of the screen in Fig. After the player selects the number of cards and wager per card, the player presses the button labelled “4 Cards Selected—Total $4.00—Buy” to initiate the game. There is no button labelled “Play”. As described in the POD brochure, “The cash display on the lower right section of the screen shows your current balance, which increases with winnings and decreases with purchases.”. Using this terminology, expenditures are not referred to as losses but rather as the purchase price of playing, and so any outcome can be presented either as a “non-win” or a “win”.When play is initiated, 24 random numbers are drawn by the computer and virtually dabbed on all cards in approximately 2 s. The 24 drawn balls are shown on the left-hand side of the screen.Lucky Clover, any winning pattern is highlighted in green . So if one were to put wagers on four cards then it would be a hit if 1, 2, 3, or 4 of the cards has a win of any size. We calculated the hit frequency broken down by cards wagered and the results are that playing 1 card has a hit frequency is 16.44 %, for 2 cards it is 20.18 %, for 3 it is 41.66 %, and over half of the outcomes when playing all 4 cards will be a win or LDW (51.25 %).Lucky Clover. The payback percentage we obtained was 91.94 %, which means an estimated hold of 9.06 %, which differs only slightly due to sampling error from the stated hold of the game which is 8.90 %  are in one physical drum, and balls are randomly drawn one at a time. The numbers on Lucky Clover are drawn randomly but from five separate arrays, as if from five different drums, with one drum for each of the five letters in B-I-N-G-O. In Lucky Clover there are always five balls drawn between 1 and 15, five between 16 and 30, four between 31 and 45, five between 46 and 60, and five between 61 and 75. The player is not informed about this difference between Lucky Clover and regular bingo. When the 24 balls are shown to the player, as in Fig. The manner in which the balls are drawn in numbered –75 are iLucky Clover and other POD Bingo games can be played at a much faster rate than paper Bingo. Players are able to complete a play in as little as 2 s, or 30 plays per minute if playing continuously. The high frequency of small wins and LDWs reinforces continued play. For example, assume a player arrives with $20 and plays until broke by betting $1.00 per play with $0.25 on each of four Lucky Clover cards. That player may lose an average of 8.90 cents per spin because the hold of the game is 8.90 %, and it may take an average of 225 spins (20 divided by $0.089) for the player to go broke. Such a session would have the player wagering a total of $225 and at 30 plays per minute it would take approximate 7.5 min for the player to go broke. The player’s original bankroll of $20 is only a small fraction of the total amount wagered. The other $205 is won mostly as LDWs and small wins, with perhaps a few larger wins. With wagers placed on four cards, the hit frequency in Lucky Clover is 51.34 %, so the player would have an average of 116 positively reinforcing hits , made up of an expected 73 regular wins and 43 LDWs—even though the player ends up going broke.Like slot machines, Lucky Clover found that players have the choice to increase the hit frequency from 16.44 % with one card played up to 51.34 % with four cards, and that approximately 19.13 % of plays result in an LDW when playing all four cards. Although LDWs are obviously losses, the celebratory sights and sounds that occur during play serve to camouflage this fact  emphasize the social benefits of fund raising. They fail to mention that the co-location of traditional paper-based Bingo beside the new electronic forms of the game, as well as slots-like video instant ticket dispensers, is potentially far more dangerous to players than the general public might realize.Bingo halls have existed in Ontario for decades and are socially accepted in large part because they provide funding to charities. In the terminology of the Pathways Model, this public acceptance is an ecological determinant that relates to “…public policy and regulatory legislation that create and foster an environment in which gambling is socially accepted, encouraged and promoted.” (p. 491). Advertisements by OLG with slogans such as “When you revitalize bingo centres in Ontario, your local charities win.” . Thus the modernization of charitable gaming in Ontario meets all three of the common processes in the Pathways Model of Problem and Pathological Gambling. Bingo players who have personal characteristics that make them susceptible to problem gambling (e.g. high impulsivity), might be more likely to realize that unfortunate potential if they partake in these more intense electronic forms of the game. We conclude that rates of problem gambling and other harms should be carefully monitored during and after the rollout of these new Bingo EGMs in Ontario, as well as in other jurisdictions that introduce this kind of gambling to their charitable or other gaming sectors. Careful monitoring is necessary to determine if future steps can be taken to minimize harm, while ensuring the safe and socially responsible modernization of these enterprises.


Animal research (AR) findings often do not translate to humans; one potential reason is the poor methodological quality of AR. We aimed to determine this quality of AR reported in critical care journals.All AR published from January to June 2012 in three high-impact critical care journals were reviewed. A case report form and instruction manual with clear definitions were created, based on published recommendations, including the ARRIVE guidelines. Data were analyzed with descriptive statistics.Seventy-seven AR publications were reviewed. Our primary outcome  was reported in 52 . Of the 77 publications, 47 (61%) reported randomization; of these, 3 (6%) reported allocation concealment, and 1 (2%) the randomization procedure. Of the 77 publications, 31 (40%) reported some type of blinding; of these, disease induction , intervention , and/or subjective outcomes  were blinded. A sample size calculation was reported in 4/77 (5%). Animal numbers were missing in the Methods section in 16 (21%) publications; when stated, the median was 32 . Extra animals used were mentioned in the Results section in 31 (40%) publications; this number was unclear in 23 (74%), and >100 for 12 (16%). When reporting most outcomes, numbers with denominators were given in 35 (45%), with no unaccounted numbers in 24 (31%), and no animals excluded from analysis in 20 (26%). Most  studies reported >40, and another 19 (25%) reported 21 to 40 statistical comparisons. Internal validity limitations were discussed in 7 (9%), and external validity (to humans) discussed in 71 (92%), most with no  or only a vague  limitation to this external validity mentioned.The reported methodological quality of AR was poor. Unless the quality of AR significantly improves, the practice may be in serious jeopardy of losing public support. Translation of biomedical animal research (AR) findings to humans has been disappointing ,2]. The. The2]. The claims made above are supported by much empirical literature. First, the poor translation rate of AR to human medicine has been found in critical care, for example, in the fields of sepsis –12], tr, tr12], For example, no novel therapy based on AR has been successful in the treatment of sepsis in humans –12]. Th. Th12]. One step in settling this debate in critical care AR is to determine the most current methodological quality of the relevant AR. To address this, we aimed to determine the reported methodological quality in critical care AR published in the year 2012. We find that the reported methodological quality of AR published in three high-impact critical care journals during 6 months of the year 2012 was poor, potentially contributing to the poor translation rate to human medicine.The University of Alberta Health Research Ethics Board waived the requirement for review because the study involved only publicly available data.Critical Care Medicine, Intensive Care Medicine, and American Journal of Respiratory and Critical Care Medicine) during 6 months of the year 2012 to determine the reporting of a priori-determined methodological quality factors. There were no restrictions other than that the study reported an AR experiment, defined as a procedure for collecting scientific data on the response to an intervention in a systematic way to maximize the chance of answering a question correctly or to provide material for the generation of new hypotheses . Ou. Ou41]. Results from the review of 77 AR publications  of publications.n = 32 . Eligibility criteria (inclusion and exclusion) for animals were reported in only 4 (5%).In the 47 61%) studies reporting randomization, the randomization method  and allocation concealment  were rarely reported ; however, age 29, 38%), developmental stage , and description of baseline characteristics in treatment groups  were often missing , with mention of negative  or toxicity  outcomes uncommon , unaccounted for , and excluded animals  were common. Extra animals were defined as follows: the number of animals used in the Results section is different from and higher than that stated in Methods section. Unaccounted for animals was defined as follows: the number of animals used in most analyses was lesser than the number given in the Methods section for unclear reasons. Excluded animals were defined as follows: animals that were stated to be used in the experiments were excluded from the majority of analyses. Outcomes were often reported without denominators in text and tables/graphs . Most studies performed >40 statistical analyses , often of tive 15, 0% or toxInternal validity limitations were rarely discussed . External validity (to humans) was mentioned in 71 (92%); however, limitations to this external validity were often not mentioned 32/71, 45%)  met the composite outcome of reporting any randomization, any blinding, and numbers given with denominators for most outcomes; only 8 (10%) met the composite outcome of the aforementioned criteria and adding meeting our primary outcome of animal descriptors Table .Funding source was reported for 69 (90%) of the publications. Most studies were funded using public dollars, either from government  and/or foundation/charity ; industry funding was uncommon .n = 27) and studies in the higher-impact journal were lower in quality . Adult animal studies more often reported sex and our primary outcome of animal descriptors (p < 0.01). The post hoc subgroup of nonrodent/nonrabbit (vs. rodent/rabbit) AR showed few differences in quality practices  to limit ventilator-induced lung injury . This map value  . Ho. Hop val. Al. Alp valthan 13% –66]. Th. Thp val. Th. Thp val likely) ,41] sug sugp valWe believe that a serious debate about the methodological quality of AR in critical care is urgent. Better attention to, and reporting of, methodological factors in AR can only improve the research quality, ethical quality, and public perception of AR, and improve the safety of humans in translational research. As we reported elsewhere, improved attention to the ethical dimension of AR can only improve these factors as well . JournalWe found that reported methodological quality of AR in three high-impact critical care journals during 6 months of the year 2012 was poor. These findings warrant the attention of clinicians, researchers, journal editors and reviewers, and funding agencies. Improved attention to the reporting of methodological quality by these groups can only improve AR quality and the public perception of AR.The authors declare that they have no competing interests.ARJ contributed to conception and design, acquisition of data, analysis and interpretation of data, and drafted the article. MB contributed to design, acquisition of data and interpretation of data, revising the article critically for important intellectual content. ARJ had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. ARJ conducted and is responsible for the data analysis. Both authors read and approved the final manuscript.Manual for the Case Report Form for Methodological Quality of animal research study. This file includes the glossary for all the variables that were abstracted from each animal research study. The definitions used are described in this file.Click here for fileCase Report Form for Methodological Quality of animal research study. This file shows the case report form used to abstract the variables from each animal research study, according to the definitions given in the manual for the case report form.Click here for fileThe publications included in the Methodological Quality of animal research study. This file includes the list of the 77 publications reviewed for this study.Click here for file


Unilateral spatial neglect (USN) is an attention deficit in the contralesional side of space which occurs after a cerebral stroke, mainly located in the right hemisphere. USN patients are disabled in all daily activities. USN is an important negative prognostic factor of functional recovery and of socio-professional reinsertion. Thus, patient rehabilitation is a major challenge. As this deficit has been described in many sensory modalities (including hearing), many sensory and poly-sensory rehabilitation methods have been proposed to USN patients. They are mainly based on visual, tactile modalities and on motor abilities. However, these methods appear to be quite task-specific and difficult to transfer to functional activities. Very few studies have focused on the hearing modality and even fewer studies have been conducted in music as a way of improving spatial attention. Therefore, more research on such retraining needs is neccessary in order to make reliable conclusions on its efficiency in long-term rehabilitation. Nevertheless, some evidence suggests that music could be a promising tool to enhance spatial attention and to rehabilitate USN patients. In fact, music is a material closely linked to space, involving common anatomical and functional networks. The present paper aims firstly at briefly reviewing the different procedures of sensory retraining proposed in USN, including auditory retraining, and their limits. Secondly, it aims to present the recent scientific evidence that makes music a good candidate for USN patients’ neuro-rehabilitation. Unilateral spatial neglect (USN) is a neuropsychological syndrome characterized by an attention deficit in the contralesional side of space . USN canA variety of clinical tests exist to assess USN. Assessment usually relies on “paper and pencil” tests. The most common conventional tests are the line bisection tasks , the canAlthough USN is essentially evaluated in the visual modality in clinical practice, deficits of spatial attention in USN have been described in many other sensory modalities: touch , hearingEven though there are only a limited number of studies on USN in modalities other than the visual modality , USN seems to affect all sensory modalities and not only vision. In the next section, we will conduct a review of the main sensory rehabilitation methods based on the theoretical conception that USN is a spatial attention deficit involving different sensory modalities. In the following sections, after underlying the contribution of hearing in USN rehabilitation, we will present the recent scientific evidence that makes music and musical practice promising tools for USN patients’ rehabilitation.The visual scanning training method  is the mOther rehabilitation methods exist and are based on the hypothesis that USN is an impairment of coordinate transformation used to represent extra-personal space and can be rehabilitated by recalibrating the perception of space. These rehabilitation techniques aim at modifying cognitive maps to induce more accurate behaviors. The cases of optokinetic stimulations or prismatic glasses in the visual modality are examples of such techniques. In other modalities, caloric or transcutaneous electrical stimulations, also rely on this hypothesis. Optokinetic stimulations are based on displays of visual stimuli all moving coherently on a computer screen to the patient’s neglected side. Some studies  showed iOther rehabilitation methods using the vestibular and the somato-sensory modalities have also been used when treating USN. These methods are based on the same hypothesis of the two previous techniques and aim to calibrate cognitive maps. Vestibular stimulation, also known as caloric stimulation, involves the use of cold water in the left ear . In thisOther approaches target motor abilities in order to help the rehabilitation of visual attention. Using motor rehabilitation , it was Finally, many rehabilitation techniques have been proposed on sensory modalities other than the visual one but difficulties in evaluating the impact of these techniqus were reported as we have previously seen. Indeed, the clinical tools used to evaluate the efficiency of a rehabilitation method are essentially based on the visual modality, and overall, do not take into account the possible improvement in other modalities and, rarely, in daily life. In order to come closer to real life improvements, studies using poly-sensory rehabilitation methods have emerged.Since we live in a multi-sensory environment, combining modalities bring the rehabilitation closer to real life. Recently, And what about hearing? In literature, very few studies take an interest in the potential effects of auditive stimulations in USN rehabilitation. This is probably due to a lack of audition assessment tools. However, some studies have provided evidence that auditory stimuli could significantly enhance visual perception in USN. These patients show an improvement in visual detection when visual and auditory stimuli come from the same position in space, unlike when they came from two different positions ,b. This Furthermore, some evidence of auditory stimulations as an effective modality training for USN patients exists. Nevertheless, contrary to all other sensory modalities, no study was really focused on the specific effect of the auditory stimulations in long-term rehabilitation. Some studies used auditory stimulations combined with other sensory rehabilitation methods. Yet, in these rehabilitation methods, auditory stimuli were only used as feed-back information or as alerts but not as a real rehabilitation tool  with an Music as an auditory stimulation is particularly interesting to examine. This is because, as we see in Recently, studies showed that just listening to music could improve spatial attention in USN . The visEven with the possibilities presented, music as auditory stimulations has not yet been used as tool for long-term rehabilitation in USN. From our review of the literature, every few studies using music material to rehabilitate USN patients was found. However, music has more frequently been used in long-term rehabilitation of patients with a cerebral stroke. The question can arise regarding how music could enhance spatial attention in USN. Could the effects of music be specific to space or be linked to more general non-spatial effects, such as those induced by emotions?The “Mozart effect” was the first piece of evidence in favor of interactions between music and space . This efListening to pleasant music can influence emotional states and activate the substrates of emotional states in the orbito-frontal cortex . AccordiAre these effects specific to music? Music could have a specific effect on the spatial attention of USN patients, not only induced by positive emotions. Space and music have close links. Some studies, carried out with healthy participants, showed that musicians had, on average, better visuo-spatial abilities than non-musicians. These studies have emphasized the idea of direct interactions between music and space. For example, the study by The same cerebral areas, located in the parietal cortex are activated by tasks comparing different musical pitches and visuo-spatial tasks. We can question why listening to musical tones can activate cerebral areas also related to space. The main considered hypothesis is that pitches can be conceptualized as positions on a mental line oriented either vertically or horizontally . This hyAccording to some authors, numbers are not the only items linked to space. The same authors also demonstrated the existence of a horizontal mental pitch line. The results were influenced by musical expertise of participants. Musicians seemed to associate automatically low-pitched tones with the left part of a horizontal line and high-pitched tones with the right part. For non-musicians, this association was found only with an explicit pitch comparison task and not with an instrumental timbre judgment task. These findings are congruent with those found by Finally, As USN patients could show impairment in the estimation of a number interval midpoint, we can ask ourselves if estimation of all magnitudes, and especially of pitches, could be affected by USN. How could these findings help to rehabilitate USN patients? Recently, Although pitch discrimination has not been studied enough in USN, evidence seems to show that music could activate spatial representations in the brain and, therefore, could be a successful tool to use in the rehabilitatation of USN.Musical practice appears to be a promising tool to rehabilitate USN for several reasons. First, playing music involves several sensory modalities: hearing, vision, touch and motor skills. Musical practice also implies higher-order cognitive processes. Musicians learn associations between motor actions, specific sound and visual stimuli and receive, in return, multisensory feedback. According to Finally, playing music could be have a greater impact than other methods. USN patients can have difficulties in being implicated in their own rehabilitation, in particular because of anosognosia . The real efficiency of musical practice has not been sufficiently examined in USN. In the literature, just one study, in which only two USN patients were included, implied musical practice . The autSeveral sensory rehabilitation techniques have been proposed for USN patients. As we saw in this review, a number of them focus only on the visual modality whereas others use motor skills, somato-sensory or vestibular stimulations to improve visual spatial attention. These rehabilitation methods are mainly based on two theoretical approaches: either recalibrating cognitive maps or retraining the orientation of spatial attention. The literature indicates that poly-sensory rehabilitation appears to be a better way to rehabilitate USN. However, all these methods present major issues concerning the generalization to daily life and the long-term efficiency.In the light of this review, both music listening (perception) and music playing (production) have been indicated as promising methods to rehabilitate spatial attention in USN patients. Music involves general effects on cognition linked to motivation and the emotions that it induces . We have seen that music could contribute more specifically to the orientation of spatial attention as music and space share closed links. Notably, musical practice could be an interesting rehabilitation tool as it also involves several sensory modalities . It is essential that more research is conducted on music and musical practice in order to determine their potential effects on spatial attention in USN patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity.Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27 Endometrial cancer (EC) is the most common gynecological malignancy. In the United States alone, 54, 870 new EC diagnoses and 10, 170 fatalities are expected in 2015. Hysterectomy with or without adjuvant treatment for localized EC yields a 5-year survival rate of approximately 95%. However, treatment outcome is poor in the 28% of EC cases that are diagnosed in an advanced stage .Recent research on the effects of androgens on endometrial tissue might have important implications for EC treatment. Polycystic ovarian syndrome (PCOS), an androgen-related disease characterized by prolonged periods of anovulation, has long been associated with increased risk of EC. PCOS and EC share many of the same risk factors, and androgens might also promote the development of EC tumors. A recent Australian study found that women with PCOS were at a four times higher risk of developing EC than women without PCOS. This risk of EC was associated with symptoms of androgen excess, such as hirsutism  and irregular periods  [The androgen receptor (AR), a nuclear transcription factor, regulates EC progression. AR can inhibit the growth of endometrial epithelial cells in culture, and this effect is blocked by an AR antagonist . Howeverin vitro and in vivo [Enzymes that regulate reversible histone lysine methylation also might play an important role in EC . One suc in vivo –12. Fina in vivo . These oin vitro and in vivo studies to identify the effects of KDM4 enzyme activity on AR signaling and EC progression. Levels of the four KDM4 proteins were decreased using siRNA in different cellular models of EC, and resulting changes in AR signaling and EC progression were measured using qRT-PCR, immunoassays, and measurements of cellular migration and proliferation. Additionally, known target genes of AR were probed in these cell lines to determine specific downstream molecular effects of manipulating KDM4 levels. Because KDM4 enzymes are important regulators of histone methylation, epigenetic changes were also examined in transfected cells. The use of cell lines with both high and low baseline AR expression AR allowed us to identify distinct roles for KDM4 proteins in EC. Xenograft experiments in which mice were injected with EC cells with either normal or reduced levels of specific KDM4 proteins confirmed their effects in vivo. Finally, tissues and genetic information from human patients with EC were examined to confirm the potential clinical relevance of our findings regarding KDM4 and AR.Here, we conducted a variety of To help establish whether AR signaling affects EC progression, we used cBioPortal to examine cross-cancer alteration summaries of AR, which included AR amplification, mutation, and deletion Figure . EC patiWe used siRNAs to decrease expression of each KDM4 methylase in order to determine whether these proteins affect AR signaling and EC cells. RT-PCR revealed that depletion of KDM4B down-regulated expression of the AR-dependent gene c-myc Figure  and 2B iTo test the effects of KDM4B knockdown on EC progression, MFE-296 cells were transfected with siRNA against AR, KDM4B, both AR and KDM4B, or a negative control (NC). Proliferation of MFE-296 cells decreased after knockdown of either AR or KDM4B. When both AR and KDM4B were knocked down, proliferation was further inhibited in MTT and clonogenic growth assays Figure  and 3B. in vivo. The final mean weight and volume of tumors in the MFE-296/shKDM4B group were significantly lower than those in the MFE-296/NC group  in AN3CA cells, which have low levels of AR Figure . KDM4B dkip1/p16/PTEN/p53) [kip1 gene  –17 aftere Figure  in respokip1 promoter in response to androgens. DHT treatment also recruited AR to the p27kip1 promoter  or NC (AN3CA/NC)  Figure . The fin) Figure . Xenogra3 Figure . Western3 Figure  and 2C.P < 0.05, **P < 0.01, Figure P < 0.05, Table Given the roles of KDM4B and KDM4A in EC cell AR signaling, expression of these two enzymes was assessed in human EC clinical material Figure . In totan = 333) in an independent dataset from TCGA network. Although UCEC patients with KDM4B and KDM4A alteration seemed to have poorer DFS rates, this effect did not reach statistical difference  of uterine corpus endometrioid carcinoma (UCEC) patients (kip1) and accelerates EC progression  signaling axis that regulates AR target genes  2008–0064). All efforts were made to minimize animal suffering.Tissue samples from 76 cases of uterine EC were obtained from patients who underwent surgery between June 1, 2011 and April 30, 2013 at the International Peace Maternity & Child Health Hospital affiliated with the Shanghai Jiao Tong University School of Medicine. The stages and histological grades of these tumors were established according to the criteria of the Federation International of Gynecology and Obstetrics (FIGO) surgical staging system (2009) . Two ind2 at 37°C.Human EC cell lines MFE-296, KLE, HEC-1B, and AN3CA were purchased from the Chinese Academy of Sciences Committee Type Culture Collection cell bank. MFE-296 cells are derived from a moderately-differentiated primary endometrial adenocarcinoma (grade 2) with high level of AR , while A5 cells/well in 6-well plates. After the cells were grown overnight, they were transfected with JMJD1C(siJMJD1C) , siKDM5A, siKDM4A, siKDM4B, siKDM4C, siKDM4D, siSMAD4, siKDM1A, siAR, or siRNA negative control (siNC)  using Lipofectamine2000  according to the manufacturer's instructions. The sequences are listed in Table 2000 (Invitrogen) according to the manufacturer's instructions.Cell transient transfections were performed as we previously reported . In brie2 [Tissue specimens of normal endometrial cells were obtained from ten women with endometriosis who had given informed consent before surgery. These women had no endometrial hyperplasia or neoplasia, and had not received any hormonal medication or anti-inflammatory drugs for at least 3 months prior to surgery. The endometrial biopsies were minced into small pieces and digested with 0.2% collagenase (Gibco). Cells were pelleted by centrifugation, resuspended in Dulbecco's modified Eagle's medium (DMEM)/F12 containing 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% FBS, and plated in 100-mm diameter culture dishes, then grown at 37°C in 5% CO2 .−7 M for different periods of time (0–24 h). Vehicle contained 0.1% absolute ethanol/phenol red–free DMEM/F12.Cells were incubated in phenol red–free DMEM/F12 (Gibco) overnight, with the final concentration of ethanol at 0.1% 5α-dihydrotestosterone (DHT)  was added to the cell culture media at concentrations of 10−7 M (100 nM) DHT and 24 h of incubation elicited the strongest expression of AR and its target genes [To investigate the impact of KDM4A and KDM4B combined with DHT treatment, cells that had been transfected with siKDM4A, siKDM4B, or siNC for 36 h were incubated with phenol red–free DMEM/F12 for 12 h. DHT or vehicle was then added according to the methods mentioned above. Previous dose-response experiments revealed that 10® realplex according to the manufacturer's instructions. Each sample was assayed in triplicate in each of three independent experiments. The primer sequences are listed in Table Total RNA was extracted from cultured cells using Trizol Reagent (Invitrogen). RNA was converted to cDNA with a one-step Prime Script RT reagent kit , and quantitative real-time polymerase chain reaction (PCR) was performed by using SYBR Premix Ex Taq (TaKaRa) in an Eppendorf Mastercyclerkip1 . Protein bands were visualized by enhanced chemiluminescence , and protein expression was normalized against β-actin .For Western blot assays, conducted as previously described , antibodTotal protein was extracted from MFE-296 cells. After protein quantification, 500 μg of each cell lysate was added to 10 μl of AR or KDM4B antibody, shaken at 4°C overnight, and then added to 30 μl of Protein A + G Agarose , shaken at 4°C for 4 h, centrifuged at 2500 × g for 5 min, and washed with a RIPA kit (Beyotime) to collect the immunoprecipitate-bound agarose beads. Each immunoprecipitate was denatured with 20 μl of 1× SDS-PAGE loading buffer at 100°C for 5 min. Each supernatant was subjected to SDS-PAGE (8% acrylamide). Other steps were as described in the Western blotting section.2000 according to the manufacturer's protocol. Cells were harvested and subjected to immunoprecipitation using AR antibody (Santa Cruz), followed by Western blotting with KDM4B antibody (Bethyl Laboratories) and KDM4A antibody.To determine whether KDM4A or KDM4B can interact with AR in AN3CA cells, AN3CA cells grown in DMEM/F12 media containing 10% fetal calf serum were transfected with 1 mg of PWP1/GFP/Neo-AR or 1 mg of its negative control PWP1/GFP/Neo after 72 h using Lipofectaminekip1 promoter between − 1523~− 1294 includes the AR binding site. Enrichment was calculated by using the comparative Ct method. Primers used (including p27kip1 promoter targeting the range − 1523~− 1294) are shown in Table Chromatin immunoprecipitation (ChIP) assays were performed as previously described , 39 usin3 cells/well in 96-well plates. Then, 20 μl of 3--2,5-diphenyltetrazolium bromide  was added to each well. Wells were subsequently incubated at 37°C for 4 h. Absorbance was measured at 490 nm using a Spectra Max 190 microplate reader . In colony formation assays, cells were seeded at 120 cells/well (MFE-296 cells) or 200 cells/well (AN3CA cells) in 6-well plates. After 14 days, cell colonies were stained with 0.1% crystal violet for 1 h and counted.In MTT assays, cells were plated at 3 × 105/well (for the migration assay) or 2 × 105/well (for the invasion assay) in invasion chambers  with or without matrigel coating for invasion or migration assays. Complete medium was added to the lower chamber as a chemoattractant. After 5 h (MFE-296) or 24 h (AN3CA) of incubation for the migration assay, or after 24 h (MFE-296) or 48 h (AN3CA) of incubation for the invasion assay, cells were fixed with 4% paraformaldehyde for 1 h. Cells on the apical side of each insert were removed by mechanical scraping. Cells that migrated to basal side of the membrane were stained with 0.5% crystal violet and counted at 200× magnification. Cells migration and invasion assays were repeated at least three times.Cells were plated serum-free at a density of 1 × 10The lentivirus vector down-expressing KDM4B was prepared for MFE-296 cells using shRNA targeting KDM4B  or a negative control vector  according to the manufacturer's protocol. The lentivirus vector down-expressing KDM4A was prepared for AN3CA cells using a shRNA targeting KDM4A  or a negative control vector  according to the manufacturer's protocol. shRNA lentiviral particles were treated in six-well plates in the presence of polybrene (6 μg/ml). Cells were treated with puromycin (2 μg/ml) to generate stable KDM4B and KDM4A knockdown clones, and several puromycin-resistant KDM4B and KDM4A knockdown clones were harvested by ring selection. The shRNA oligo sequences are provided in Table 6 cells of one of the following cell types: MFE-296/shKDM4B, MFE-296/shNC, AN3CA/shKDM4A, or AN3CA/shNC. Tumor size was monitored every 4 days by measuring the length and width with calipers, and tumor volumes were calculated with the formula: tumor volume (cm3) = (the longest diameter) × (the shortest diameter)2 × 0.5. The tumors were removed and weighed after 42 days (for the two MFE-296 groups) or after 49 days (for the two AN3CA groups).For the xenograft experiments, 20 5-week-old female BALB/c nude mice  were randomly divided into four groups. The mice were injected subcutaneously in the nape with 5 × 10Paraffin-embedded normal endometrial, atypical hyperplasias, and EC tissue sections (4 μm) were processed for immunohistochemical analysis as previously described . Brieflykip1 antibody , and ki-67 antibody  were used.In addition, tissue specimens (4 μm) of mouse xenografts were routinely prepared and immunohistochemically analyzed by using the standard avidin-biotin techniques. KDM4B antibody, c-myc antibody , KDM4A antibody, p27http://cbioportal.org) provides a Web resource for exploring, visualizing, and analyzing multidimensional cancer genomics data. Specifically, because of interests in the gene set of AR, we entered AR gene symbols, and then a report of genomic data across all cancer types was presented in a concise graphical summary called a cancer genomics of cross-cancer alteration summary of AR. We then selected endometrial carcinoma, entered AR and KDM4B gene symbols, and a network view of the AR neighborhood in EC was shown. All genes were ranked by the frequency of genomic alteration within the EC study, and less frequently altered genes were automatically pruned from the network.The cBioPortal for Cancer Genomics /disease-free survival (DFS) of EC patients using published data from The Cancer Genome Atlas (TCGA) network [P values from a log-rank test.We performed an  network , 44. Clit test was used for comparison between two groups, and one-way ANOVA followed by the post-hoc Tukey test were used for comparison among multiple groups. Correlation was analyzed by the Spearman test, and OS was assessed by using the standard log-rank test and the Kaplan-Meier method. All P values were two-tailed, and P values < 0.05 indicated a statistically significant difference. Each experiment was performed at least three times independently.Data were presented as mean ± SD and analyzed by SPSS 16.0 software . The Student