SH2B2) gene regulate energy balance and body weight at least partially by enhancing Janus kinase-2 (JAK2)-mediated cytokine signaling, including leptin and/or GH signaling. Leptin is an adipose hormone that controls body weight. The objective of the present study is to evaluate the association between body measurement traits and SH2B2 gene polymorphisms as responsible mutations. For this purpose, we selected four single-nucleotide polymorphisms (SNPs) in SH2B2 gene, including two in intron 5 . Association study indicated that the g.20570G>A, g.20693T>C, and g.24070C>A, significantly (P < 0.05) associated with body length (BL) and chest circumference (CC) in Qinchuan cattle. In addition, H4H3 and H5H5 diplotype had highly significantly (P < 0.01) greater body length (BL), rump length (RL), and chest circumference (CC) than H4H2. Our investigation will not only extend the spectrum of genetic variation of bovine SH2B2 gene, but also provide useful information for the marker assisted selection in beef cattle breeding program.The Src homology 2 B 2 ( MarkeSH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. Previously, SH2B2 that categorized as an adapter protein with a PH and SH2 domain (APS) is a member of the Src homology 2 B (SH2B) and has a conserved structure of a N-terminal dimerization domain (DD), a central pleckstrin homology (PH) domain, and a C-terminal Src homology 2 (SH2) domain [SH2B2 may regulate energy balance and body weight partially by enhancing Janus kinase-2 (JAK2)-mediated cytokine signaling, including leptin and growth hormone signaling. In cultured cells, SH2B2 binds via its SH2 domain to JAK2, potentiating JAK2 activation [. Moreover, SH2B2 didn’t affect insulin receptor numbers or insulin receptor turnover both in vivo and in vitro; however, SH2B2 increased insulin sensitivity in mice [SH2B2 has activity in mediating the insulin-stimulated activation of the c-Cb1/CAP/TC10 pathway that appears to play an important role in regulating glucose uptake in cultured adipocytes [SH2B2 is expressed in multiple tissues, including targets of insulin, GH, and leptin  [SH2B2, on the other hand, are known as negative regulators of B-cell proliferation [SH2B2 not only in fat but also in kidney and other splanchnic tissues, which might bring some change about animal traits. Thus, we hypothesized that SH2B2 might be associated with conformation and carcass traits on beef cattle.The SH2B family has three members  domain . SH2B2 mtivation ,16 and ativation ,18. More in mice . Therefoipocytes . SH2B2 i muscle) ,21,22. Sferation ,24 and tSH2B2 genotypes with body measurement traits in Qinchuan cattle. Therefore, the present study was designed to identify the effects of polymorphisms on SH2B2 in 468 individual Chinese Qinchuan cattle by using Real-time PCR to analyze tissue expression patterns and establishing a correlation between the bovine SH2B2 gene mutations and body measurements to identify associated quantitative traits for the benefit of cattle breeding and genetics.There has been a lack of information about the association of bovine SH2B2 protein in different species. The amino acid sequences of SH2B2 gene were acquired from NCBI (www.ncbi.nlm.nih.gov/protein) for Bos taurus (XP_024841048.1), Homo sapiens (NP_066189.3), Ovis aries (XP_027817506.1), Mus musculus (NP_061295.2), Ovis aries (XP_023511156.1), Bubalus bubalis (NP_001277771.1), Equus caballus (NP_001265704.1), Gallus gallus (XP_015151487.1), Felis catus (XP_023102534.1), Cavia porcellus (XP_013008193.1) Oryctolagus cuniculus (XP_008251155.1) Macaca mulatta (XP_014990031.1) Canis lupus familiaris (XP_005621054.1). Sequence similarity between bovine SH2B2 protein and its homologue was performed. The Jalview Jalview software’s were used for multiple sequence alignment . The analysis of protein structure and function, the motifs were searched, and conserved domains were identified through the online MEME suite website [The bioinformatics techniques were used for the measurement of degree of conservation and biological evolution of  XP_0231034.1, CavQinchuan cattle breeding populations, the subject animals were fed a total mixed ration (TMR), containing 25% concentrate and 75% roughages of dry straw and corn silage, and water was offered ad libitum. The feeding was offered based on NRC standards (Nutrient Requirement of Beef Cattle) [Total 468 female cows (non-pregnant) of Qinchuan breed cattle maintained at the experimental farm of National Beef Cattle Improvement Research Centre, Yangling, China were selected for conducting this research study. All the experimental animals were aged between 18 and 24 months of age and were randomly selected from  Cattle) . MoreoveGenomic DNA were extracted from blood samples (collected from the jugular vein) using a blood DNA Kit . The DNA content was estimated spectrophotometrically, and diluted to 50 ng/μl. Meanwhile, body measurement traits (BMTs), including body length (BL), withers height (WH), hip height (HH), rump length (RL), hip width (HW), chest depth (CD), and chest circumference (CC), were measured as described previously  for assoSH2B2 gene were designed based on NCBI database (GenBank accession number NC_037352.1) CDS region of ∼1949 kb. First, we mixed 468 DNA samples with equal molar ratio to constitute a DNA pool [2 and 0.5 U Taq DNA polymerase . The PCR protocol was 5 min at 95°C; 35 cycles of 30 s at 94°C, 35 s at corresponding temperature, 40 s at 72°C, and a final extension step at 72°C for 10 min. The digested products were detected by electrophoresis technique in a 0.8% agarose gels containing 0.5 μg of ethidium bromide/ml. The PCR products were sequenced through Sangon  to screen for polymorphisms. All sequences were checked using Seq Man  software, and the SNPs were identified.There primers to amplify of the bovine DNA pool . Then, Dn = 3). The RNA was extracted from each tissue sample using the Trizol reagent kit , and was subjected to reverse transcription (RT) to obtain the corresponding cDNA . After collection from the tissue, samples were preserved in liquid nitrogen and were transferred immediately in frozen form to the molecular laboratory for the extraction of total RNA. The total RNA was extracted from the tissue using TRIzol™ Reagent . Data were normalized to the geometric mean of GAPDH (GenBank Accession no. NM_001034034) used as endogenous control genes. The primers used are given in The eight tissue specimens, including muscle, rumen, fat, abomasum, heart, spleen, kidney, and small intestine were collected from three female Qinchuan cattle aged 18 months old  were calculated through χ2 test via the PopGene software . LinkageAnalysis of associations between the genotypes of SNPs and body measurement traits was carried out with the GLM procedure, using SPSS software (version 13.0) by the following formula: Yij = u+Gi+Ai+EijkWhere Yij was the traits measured on each of the individual cattle, μ was the overall population mean for the traits, Gi was the fixed effect associated with the genotype, Ai was the fixed effect due to the age and Eijk was the standard error.The mean relative mRNA expression level of SH2B2 gene in different tissues and at different age groups was analyzed by ANOVA using computer software SAS (version 8.1).2 test, the genotypic distributions of g.20570G>A, and g.20693T>C, differed significantly from Hardy–Weinberg equilibrium (P < 0.05)  see in . GeneticD and other is r2. There is a consensus of the researchers that the latter indicator is most commonly used for pair wise measurement of the LD and hence consider less sensitive for the measurement of allelic frequencies than D’ [There are two most commonly used indicators for the prediction of linkage disequilibrium (LD). One is  than D’ ,34.In the present study, the LD was highest between g.20693T>C, and g.24070C>A, . In addiP < 0.05). At g.20693T>C locus, genotype CC had higher mean values for BL and CC than these with the genotype TT (P < 0.05). At g.24070C>A, locus, significant differences of BL, RL and CC were observed between CC and AA genotypes (P < 0.05). No significant correlations were observed in the rest of the index for the four SNPs. In 4H3 and H5H5 diplotype had highly significantly greater BL, RL and CC than H4H2 (P < 0.05), similarly results were found between H4H3 and H1H1 (P < 0.05).In this paper, four polymorphisms seem to mainly affect bovine body measurement traits . At g.20SH2B2 relative expression levels in each tissue were shown in SH2B2 was expressed only slightly in the heart and kidney tissue. There could be both direct and indirect relationships between body size and metabolism due physiological modulation from SH2B2. We also analyzed expression level of SH2B2 in bovine preadipocytes and adipocytes at different time points (SH2B2 in differentiated adipocytes was decreased in day-2 (D2) as compared with day-0 (D0) of preadipocytes. Interestingly, we found an increasing trend in the expression level of SH2B2 from D2 to day-10 of adipocytes differentiation.Results for e points B. ExpresSH2B2 is 25296 bp, comprising the genomic coordinates starting from 34677735 to 34703030 . This gene comprises 11 exons, the ORF which started from the start codon to the stop codon is 2040 bp, and the putative protein contains 679 amino acids  that were detected in the bovine SH2B2 gene coding sequence (CDS) region possibly affects body measurement traits (BMTs) and meat quality traits (MQTs). To reveal the linkage relationships among these four SNPs, the linkage disequilibrium (LD) between these four sites were estimated, which indicated that the e strong  Our resuIn the present study, we found significant associations of genotypes g.20570G>A and g.24070C>A, with body measurement and carcass quality traits. Here, both g.20570G>A and g.24070C>A were located in the intron region and did not change the structure of the encoded proteins, but our results demonstrated that it was still associated with several growth traits. Such associations may be the result of linkage disequilibrium between this SNP and other genes on the same chromosome that have a significant effect on the growth traits studied here . AnotherThus, we further analyzed the effects of the combined genotypes above and growth traits in cattle. Haplotypes composed of SNPs could provide accurate information than single marker analysis for economic trait associations, due to the ancestral structure captured in the distribution of haplotypes. The Hap1 (–AATC–) had the highest haplotype frequencies (33.70%). The probable cause could be artificial selection in the Qinchuan cattle population, particularly the genomic regions influencing traits of economic importance ,45SH2B2 gene in the growth and development of Qinchuan cattle, mRNA expression was investigated in different tissues and adipocytes of Qinchuan cattle. Highest expression was found in small intestine, muscle, and fat. These findings show the role of SH2B2 in metabolism, growth and development, which are supported by the previously published literature, and that SH2B2 is a positive regulator of energy and glucose metabolism [Moreover, to further exploit the function of the tabolism . In additabolism . Similartabolism .P < 0.01) greater body length (BL), rump length (RL), and chest circumference (CC) than H4H2. Our investigation will not only extend the spectrum of genetic variation of bovine SH2B2 gene, but also provide useful information for the marker assisted selection in beef cattle breeding program.In conclusion, association analysis between SH2B2 gene polymorphisms indicated that g.20570G>A, g.20693T>C, and g.24070C>A, significantly associated with growth traits in Qinchuan cattle. In addition, H4H3 and H5H5 diplotype had highly significantly (


Using ChIP-seq and functional genomic analyses, the study shows that the Paf1 complex occupies transcriptional enhancers and positively regulates their activity. The RNA polymerase II (RNAPII) associated factor 1 complex (Paf1C) plays critical roles in modulating the release of paused RNAPII into productive elongation. However, regulation of Paf1C-mediated promoter-proximal pausing is complex and context dependent. In fact, in cancer cell lines, opposing models of Paf1Cs’ role in RNAPII pause-release control have been proposed. Here, we show that the Paf1C positively regulates enhancer activity in mouse embryonic stem cells. In particular, our analyses reveal extensive Paf1C occupancy and function at super enhancers. Importantly, Paf1C occupancy correlates with the strength of enhancer activity, improving the predictive power to classify enhancers in genomic sequences. Depletion of Paf1C attenuates the expression of genes regulated by targeted enhancers and affects RNAPII Ser2 phosphorylation at the binding sites, suggesting that Paf1C-mediated positive regulation of pluripotency enhancers is crucial to maintain mouse embryonic stem cell self-renewal. STARR-seq has been applied genome-wide in flies and in some mammalian cells and has greatly advanced our understanding of how enhancer activities are encoded in the genome  has been identified as a crucial checkpoint for RNAPII promoter-proximal pausing and pause-release at both protein-coding genes and at enhancers. However, divergent effects during promoter-proximal pausing and pause-release of Paf1C have been reported in different cancer cell lines , 15, 16.Our group has previously identified Paf1C as an important factor to maintain mouse embryonic stem cells (mESCs) pluripotency by specifically regulating the expression of pluripotency genes , 20. PafOct4, Sox2, Nanog, Esrrb, and Zfp42 in mESCs  were only bound by Ctr9 in NIH3T3 cells , suggesting that after initiation, RNAPII is paused at the proximal promoters by NELF. With the decrease in NELFA and RNAPII Ser5p, Ctr9 piled up and reached its maximum level around 145 bps downstream of the RNAPII Ser5p peaks (184 bps downstream of the TSS) . These dOct4 enhancers (P < 2.2 × 10−16) than on TEs . He. HeOct4 nhancers , Paf1C bnhancers . 72% SEsnhancers , 30. Furn on TEs .H3K4me1 and H3k27ac modifications and Ctr9 occupancy at super enhancers and typical enhancers in mouse embryonic stem cells.Table S3 Oct4, Nanog, Sox2, Esrrb, and Tbx3. In contrast, only 35 of the mESC SEs (15%) were also bound by Ctr9 in NIH3T3 cells (Table S4). Many of these common Ctr9-bound SEs were found in the vicinity of genes regulating fundamental biological processes, such as metabolism , transportation (Slc6a6 and Ssr2), scaffold (Mesd and Spry2), stress response (Nfkbia and Gadd45a), and RNA processing (Gbx2 and Gtf3c6), suggesting that these SEs serve a similar function in mESCs and NIH3T3 cells.SEs associate with many biological processes, which define cell identity, and exhibit strong cell type specificity . We therNELF, RNAPII Ser2p, RNAPII Ser5p occupancy, and enhancer RNAs expression at super enhancers in mouse embryonic stem cells.Table S4 H3K4me1 is thought to mark active enhancers, and more prominently poised enhancers, whereas H3K27ac is believed to mark active enhancers only . To deteP < 4.89 × 10−7), suggesting that more active enhancers are bound by Ctr9  and those without Ctr9 binding (SE/Ctr9−), we cloned 21 randomly selected SEs with Ctr9 binding and 28 SEs without Ctr9 binding into a plasmid that reports enhancer activity when transfected into mammalian cells. Importantly, this assay has previously demonstrated to faithfully reflect in vivo enhancer activity , 34. TheRegions of super enhancers with/without Ctr9 occupancy, and primers for luciferase reporter plasmid cloning for Fig 2G.Table S5 The Ctr9 occupancy at active enhancers encouraged us to check whether other components of the transcription apparatus associate with Paf1C to regulate enhancer activities on these sites. We first analyzed NELFA enrichment at SEs. 119 NELFA binding sites were detected for the 166 SEs (71%) that showed Ctr9 binding, suggesting that most active SEs are regulated by both Paf1C and NELF complexes . InteresPaf1C has been shown to associate with RNAPII Ser5p and Ser2p at promoters and the coding regions of genes . HoweverBulk accumulation of Ctr9 and RNAPII Ser2p at enhancers that correlated with high enhancer activities prompted us to inspect in situ enhancer transcription. We aligned our Ctr9 and RNAPII Ser2p ChIP-seq data with a published global run-on sequencing (GRO-seq) data set, which can be applied to detect eRNA transcripts in mESCs . AnalysiTo investigate whether Paf1C directly regulates enhancer activity in mESCs, we performed Ctr9 knockdown experiments . 24 h poRegions of super enhancers with/without Ctr9 occupancy, and primers for luciferase reporter plasmid cloning for Fig 5A and B.Table S6 Expression changes of genes associated with super enhancers, typical enhancers, and other genes after Ctr9 depletion, resource data for Fig 5C (three worksheets).Table S7 Oct4 and Tbx3  in this cell type. To test our prediction experimentally, we selected eight sites of H3K27ac+/Ctr9− and four sites of H3K27ac+/Ctr9+ in both mESCs and NIH3T3 cells, and eight sites of H3K27ac+/Ctr9+ only in NIH3T3 cells. The enhancer activity was determined again using the luciferase reporter system in the respective cell lines. All four sites of H3K27ac+/Ctr9+ in mESCs and NIH3T3 cells showed marked enhancer activity in both cell lines, whereas seven out of eight H3K27ac+/Ctr9+ NIH3T3-specific regions exhibited prominent enhancer activities only in NIH3T3 cells. Furthermore, all eight H3K27ac+/Ctr9 regions were inactive in both cell types  and NIH3T3 cells were cultured in Glasgow Minimum Essential Medium , supplemented with 10% FBS , 1,000 U/ml LIF , 100 mM nonessential amino acids , 2 mM L-glutamine , and 50 μM 2-mercaptoethanol .Endoribonuclease-prepared siRNAs (EsiRNAs) were produced for gene knockdown as described previously . EsiRNA Primers for esiRNA production, qRT-PCR, short guide RNA cloning, and Ctr9-GFP knock-in donor construct.Table S10 Short guide RNA and donor repair template construct (GFP with 800 bp homologous arms on both sides to Ctr9 C-terminus cutting site) were cloned as previous reported . OligonuEnhancer fragments were cloned into the KpnI site of the pGL4.23 vector. mESCs and NIH3T3 were transfected with pGL4.23 enhancer reporter plasmids containing designated sequences using Lipofectamine 2000 (Invitrogen). The pRL-SV40 plasmid (Promega) was co-transfected as a normalization control. Cells were incubated for 24 h, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). For knockdown experiments, esiRNA was transfected into mESCs using Lipofectamine 2000. mESCs were fed with fresh medium and transfected with pGL4.23 enhancer reporter plasmid and pRL-SV40 using Lipofectamine 2000. The genomic coordinates of the cloned fragments and the primers used are listed in Table S10.ChIP-seq experiments were performed as previously described with minor modification . 10 millhttps://github.com/Boyle-Lab/Blacklist. The number of sequenced fragments aligning to each base was normalized to the total number of reads in a sample. This normalization step was performed automatically by the peak calling software. Peak calling was performed using macs2 (ver. 2.2.6) , using standard methods from tidyverse packages   using bo. 2.2.6) , using a. 2.2.6)  and BEDT. 2.2.6) . Heat mapackages . Enhance18 primer. qPCR quantification of gene knockdown was performed with the SYBR Green qPCR kit (Abgene) with primers as shown in Table S10.Total RNA was extracted from cells using RNeasy Mini Kit (QIAGEN). 500 ng total RNA was reversed transcribed with Superscript III Reverse Transcriptase (Invitrogen) using an oligo d(T)RNA-Seq was performed on an Illumina HiSeq 2000, aiming for ∼30 million sequencing reads per sample. Expression levels of individual transcripts were estimated by kallisto (ver. 0.46.1)  using GEImmunostaining was performed as previous reported . Briefly5 mES cells  were reverse transfected with 1,000 ng esiRNAs and 2 μl lipofectamine 2000 in fibronectin-coated six-well plates. 72 h post transfection, ESCs cells were harvested and lysed in Laemmli sample buffer. 10 μg of protein extracts were separated on NuPAGE 4–12% Bis-tris protein gels (Invitrogen) and blotted to nitrocellulose membrane (Millipore). The membranes were probed with the primary antibodies against anti-GFP (MPI-CBG), and Tubulin , and corresponding secondary antibodies . The membranes were scanned by an Odyssey Infrared Imager, and the proteins were quantified by the software Image Studio.1.5 × 10http://ncbi.nlm.nih.gov/geo) with accession number GSE149999.The supporting data sets including ChIP-seqs and RNAseqs have been deposited in the Gene Expression Omnibus database (


CDKN2A/B locus contains crucial tumor suppressors and a lncRNA gene ANRIL. However, the mechanisms that coordinately regulate their expression levels are not clear.The CDKN2A gene were screened by CDKN2A-specific RNA capture deep-sequencing and confirmed by Northern blotting and clone-sequencing. Long non-coding RNA (lncRNA) binding proteins were characterized by RNA pull-down combined with mass spectrometry and RNA immunoprecipitation. LncRNA functions in human cells were studied using a set of biological assays in vitro and in vivo.Novel RNAs transcribed from the P14AS with its promoter in the antisense strand of the fragment near CDKN2A exon 1b in human cells. The mature P14AS is a three-exon linear cytoplasmic lncRNA (1043-nt), including an AU-rich element (ARE) in exon 1. P14AS decreases AUF1-ANRIL/P16 RNA interaction and then increases ANRIL/P16 expression by competitively binding to AUF1 P37 and P40 isoforms. Interestingly, P14AS significantly promoted the proliferation of cancer cells and tumor formation in NOD-SCID mice in a P16-independent pattern. Moreover, in human colon cancer tissues, the expression levels of P14AS and ANRIL lncRNAs were significantly upregulated compared with the paired normal tissues.We characterized a novel lncRNA, P14AS, transcribed from the antisense strand of the CDKN2A/P14 gene, promotes colon cancer development by cis upregulating the expression of oncogenic ANRIL.A novel lncRNA,  P16INK4A, P14ARF and P15INK4B) and the oncogenic lncRNA ANRIL (CDKN2B-AS1) resides in the human CDKN2A/B locus at chromosome 9p21 [P15 and/or P16 allows cells to escape cell cycle arrest in G1 while P14 protein binds to MDM2 and results in P53 activation. While ANRIL was reported to downregulate P15 and P16 expression by interacting with components of polycomb repressive complex-1/-2 [ANRIL was also found to be coordinately transcribed with P16 in cancer cells and transcriptionally repressed by P16 DNA methylation [Three important tumor suppressor genes  to identify given and novel RNAs from this gene and flanking regions. We characterized, for the first time, a novel lncRNA called P14AS (NCBI GenBank MK574077) transcribed from the antisense strand of the fragment around exon 1β of the CDKN2A gene. We found that AUF1 binds to P14AS and the AUF1 binding of the ARE (AU-rich element) in exon 1 of the P14AS gene increases ANRIL/P16 level. Furthermore, a significantly higher level of P14AS exists in colon cancers compared to paired normal tissues and that P14AS could markedly promote the proliferation of cancer cells and tumor formation in a P16-independent pattern in vitro and in vivo.AUF1 is an essential RNA binding protein that promotes the decay of many cancer-related RNAs, including C, NEAT1 –10. In tThe human cell line HEK293T was kindly provided by Professor Yasuhito Yuasa at Tokyo Medical and Dental University. HCT116 and SW480 cells were kindly provided by Professor Yuanjia Chen at Peking Union Medical College Hospital. The MCF7 cell line was kindly provided by Professor Yuntao Xie; BGC823, MGC803 and SGC7901 cells were kindly provided by Professor Yang Ke; A549 and HEK293FT cells were kindly provided by Professor Zhiqian Zhang; the HepG2 cell line was kindly provided by Professor Qingyun Zhang; the AGS cell line was kindly provided by Professor Chengchao Shou at Peking University Cancer Hospital and Institute.N = 172, including 75 cases in the pilot study and 97 additional cases in the clinical association analysis), and normal colon mucosa biopsy samples from noncancer patients (N = 50) were collected and stored at − 70 °C at Peking University Cancer Hospital from 2004 to 2011. Research protocols were approved by the Institutional Review Board of the Peking University Cancer Hospital and Institute, China. Clinical and histopathological data for each patient were obtained according to approved institutional guidelines.The colon cancer (CC) tissues, paired normal tissues from the surgical margin  from CC patients . In total, 3 μg of cDNA was fragmented by nebulization. The fragmented DNA was repaired, and an ‘A’ was ligated to the 3′ end. Illumina adapters were then ligated to the fragments, and the sample was size-selected with a 350–400-bp product. The size-selected product was amplified by PCR. Each sample was tagged with a unique index during this procedure, and the final product was validated using a Bioanalyzer . The amplified DNA 1 μg) was mixed with a  μg was mIllumina clean reads were aligned to each human reference genome using the BWA program, and quality scores were recalibrated and realigned to references using the GATK software package. Duplicated reads were removed using Sequence Alignment/Map tools (SAMtools), and only uniquely mapping reads were used for variation detection. The RNA-seq datasets were uploaded into the NCBI website (GES128205).32P-labeled RNA probes . The RNA 10 μg) was transferred to a positively charged NC film with 20× SSC buffer . The membrane was UV-cross-linked and incubated with 0 μg was P14AS lentiviral vector was constructed in PCDH-CMV-EF1a-copGFP-T2A- Puro lentiviral vector by Syngentech Co., Ltd. . The lentiviruses for the empty control and P14AS expression vector were generated with the lentiviral packaging kit  according to the manufacturer’s manual. Briefly, HEK293FT cells were seeded in 6 cm diameter plates, and transfected with vectors at 40% confluence. The medium was collected after 48 h, and filtered with a 0.45 μm filter. The stably transfected HCT116, SW480 and MGC803 cells were selected for 3 days after infection of these viruses with 1 μg/mL puromycin .The 1043-nt The four AUF1 isoforms  in pcDNA3.1 vector were kindly provided by Professor Xiaotian Zhang at Beijing Normal University . To consAUF1 gene was knocked down in stably P14AS-overexpressed HCT116 cells for 72 h. The transcriptomes of these cells were sequenced, as described in the supplemental method  [The ols.jsp) .P14AS gene and its promoter by the CRISPR/Cas9 system, respectively. The oligonucleotides used for sgRNA construction were individually designed upstream and downstream of the target fragment (Table P14AS ARE−/promoter-KO-negative clones were pooled and served as a wildtype (WT) control.A dual gRNA approach was used to knock out the ARE-containing sequence in exon 1 of the P14AS probe #1-#6, and control probe #1-#2 (Escherichia coli strain genome)  according to the manufacturer’s instructions. Total AUF1-binding RNAs were immunoprecipitated and extracted using AUF1 antibody . cDNA was synthesized from the RIP-RNAs using random primers, and gene-specific quantitative PCR was then performed using the primer P14AS expression vector or P14AS knockout vector were harvested by trypsinization, washed twice with PBS, and then subcutaneously injected into the bilateral inguinal area of female NOD/SCID mice . On the 17th posttransplantation day, mice were sacrificed. For the experiment on tumor growth from P14AS−/− and P14AS+/+ HCT116 cells (2 × 106 cells per injection), the mice were sacrificed on the 19th posttransplantation day. Tumors were fixed in 4% paraformaldehyde, sectioned, and stained with hematoxylin and eosin (H&E).HCT116 cells stably transfected with the control, P < 0.05 (*) or P < 0.01 (**).All statistical analyses were performed using SPSS 18.0 software. The Kolmogorov–Smirnov test was used to estimate the normality of distributions. The Mann–Whitney U-test was conducted for non-normally distributed data. Student’s t-test was conducted for normally distributed data. All statistical tests were two-sided. Statistical significance was assigned at P16 CpG islands, were listed in the supplemental method , and induction of methylation of CDKN2A-specific RNA transcripts in HEK293T and MCF7 cells  and AGS .An RNACap-Seq technology was established and used to screen novel lls Fig. a. A threANRIL, are often processed into circular RNAs. To study whether P14AS is a circular RNA, the linear RNAs were digested by RNase R before cDNA synthesis by reverse transcription (RT). No RT-PCR product was amplified from the RNase R-digested samples, indicating that P14AS is a linear lncRNA  in the human CDKN2A locus. Also, P14 and P16 mRNAs share the same CDKN2A exon 2 with different translation reading frames  completely overlaps with the 5′-sequence in exon 1 of ANRIL , we performed the RNA-Immunoprecipitation (RIP) assay and found that P14AS was significantly enriched in the AUF1 antibody-RIP complexes compared with the IgG control  could inhibit most of the P14AS-binding to AUF1 P37 and P40. In addition, there were two P14AS-binding bands for the AUF1 P42 and P45 complexes and unlabeled P14AS probe could only inhibit the P14AS-binding to one of these AUF1 bands. These results suggest specific bindings between P14AS and AUF1, at least for P37 and P40 isoforms.We performed a biotin-labeled RNA pull-down and mass spectrometry assay to identify potential cis regulators of nearby genes, we determined whether P14AS could affect the expression of ANRIL and other neighboring genes. The qRT-PCR results revealed that stable P14AS overexpression consistently increased the expression level of ANRIL in SW480, HCT116, and MGC803 cells  in the HEK293T (KO-1 and -2) and HCT116 clones (KO-1 and -2)  while AUF1 knockdown upregulated many lncRNA genes (N = 341). However, after the AUF1 knockdown, P14AS could not upregulate ANRIL expression . No significant changes in P15 and P16 mRNA levels were observed. These results imply that P14AS could protect the decay of lncRNAs, including ANRIL, in an AUF1-dependent pattern. The results of David functional annotation analyses showed that about half of P14AS-upregulated and -downregulated genes were significantly enriched with sequences containing signal peptide feature or related to glycoproteins  project [R = 0.23 (or 0.30 for 224 cell lines with relative CDKN2A copy number > 0), P < 0.001; Additional file AUF1 expression did not correlate with that of P16 and inversely correlated with P15 expression in these cell lines. Overall, these results suggest that P14AS could upregulate the expression of ANRIL (and other lncRNAs), probably in an AUF1 binding-dependent pattern.Interestingly, a positive relationship between the expression levels of P14AS overexpression on cancer cell behavior in vitro and in vivo. Both human colon cell lines SW480 and HCT116 and gastric cancer cell line MGC803 were stably transfected with the P14AS expression vector. The results of CCK-8 and long-term dynamic observation assays revealed that P14AS overexpression promoted cell proliferation but did not promote cell migration  controls  were injected with HCT116 cells stably transfected with P14AS into the left inguinal area and the empty control vector into the right inguinal area for each mouse. On the 17th posttransplantation day, the number and weight of tumors in the P14AS group were significantly higher than those in the control group  and their corresponding surgical margin (SM) tissue samples from 172 patients, and normal colon mucosa biopsy samples from 50 noncancer patients. The results of qRT-PCR detection (254-bp) revealed that the average P14AS level in CC tissues, with and without ANRIL expression, was significantly higher than that in the paired SM tissues and normal biopsies  was significantly higher in CCs than SMs  was significantly higher in CCs than SMs .In addition, the positive rate of P16 mRNA level (by qRT-PCR) was also significantly higher in the P14AS-positive colon CC and SM tissues  than in the P14AS-negative tissues . A similar difference was also observed between CCs with and without ANRIL expression  with CRISPR/Cas9 and found that the level of P14AS transcription was dramatically decreased in two HEK293T subclones  was significantly reduced . It also hosts the oncogenic antisense lncRNA gene ANRIL, which is implicated in cancers of the colon, breast, lung, and bladder [P14AS, in this locus. Our systemic studies show that P14AS cis upregulates ANRIL and promotes cancer cell proliferation.Accumulating evidence has revealed that lncRNAs function in multiple cellular processes, including transcription, intracellular trafficking, chromosome remodeling, and disease development. Deregulation of lncRNAs was found in various cancers –16. The  bladder –22. In tP14AS was frequently overexpressed in CC tissues, and that P14AS overexpression promoted the proliferation of cancer cells. In contrast, P14AS knockdown by siRNA and knockout of the ARE-containing element in P14AS exon 1 by CRISPR/Cas9 could repress cancer cell proliferation. The effect of P14AS on cancer cell proliferation was confirmed in a mouse model. These results suggest that P14AS may be an oncogene. We also analyzed the expression status of P14AS in gastric carcinoma tissues (N = 40), P14AS expression was not detected (data not shown). This result warrants studying its roles in cancer development in other organs.We found that P14AS could upregulate P16 expression in cell lines and P14AS expression is positively correlated with P16 expression in colon tissues, P14AS overexpression or knockout remains to affect the proliferation of the P16-inactive cell lines (HCT116 and SW480), suggesting that P14AS may function as an oncogene in a P16-independent way. P16 is a weakly expressed nucleic protein in normal cells. However, the P16 protein is sequestered in the cytoplasm without tumor suppressor function in many cancer tissues, including CCs [P16 expression by P14AS in cancers may not affect cancer development. Instead, dysfunctions of P16, P14, and P15 cause abnormal G1-S shift in the cell cycle that plays crucial role in the malignant transformation of human cells due to increased phosphorylation of RB1 [In addition, although n of RB1 .c-MYC and P16 mRNA stability [P16 mRNA, thereby influencing cell senescence [NEAT1 (10) and linc-ROR [P14AS exon 1 that binds to AUF1. Notably, knockout of the ARE-containing element not only abolishes the function of P14AS to upregulate ANRIL and P16/P14/P15 expression but also eliminates the effect of P14AS on cancer cell proliferation. Because re-expression of the wild-type P14AS could restore the effect of P14AS-KO on cell proliferation and stable knockdown of AUF1 expression could abolish the effect of P14AS on cell proliferation, P14AS-AUF1 binding is highly considered to be the main pathway for the function of P14AS.AUF1 is a member of the family of RNA binding proteins that is termed ARE/poly (U)-binding/ degradation factor 1. The canonical functions of AUF1 are to regulate mRNA decay and translation via recognition of specific sequence elements in mRNA 3′ untranslated regions (3’UTR) . For exatability , 8. Prevnescence . Moreovelinc-ROR . InteresP14AS-binding to the AUF1 P37 and P40 band was inhibited by the unlabeled P14AS probe (× 10), suggesting high binding specificity between these AUF1 isoforms to P14AS.It has been reported that AUF1 isoforms  could form dimers, which could yield oligomeric AUF1 complexes , 26. In P14AS overexpression and knockout/knockdown significantly increased and decreased ANRIL expression in many cell lines, respectively. The results of our RNA-seq analyses confirmed the upregulation (fold change > 2.5) of ANRIL expression by P14AS in HCT116 cells. Both P14AS and ANRIL were upregulated in CC tissues, and more P14AS was detected in the ANRIL-positive tissues than the ANRIL-negative tissues. In addition, the results of our AUF1 RIP-PCR experiment indicate that ANRIL lncRNA is an AUF1-binding RNA. Analysis of the public CCLE data reveals that the AUF1 mRNA level was positively and significantly correlated with the ANRIL lncRNA level in 1037 human cancer cell lines. The level of AUF1 mRNA was positively and significantly associated with the P14AS level in CC tissues. These findings support that P14AS may function as an oncogene through the cis upregulation of ANRIL expression in cancer cells through AUF1-P14AS binding.Moreover, we found that AUF1 knockdown, P14AS downregulated the expression level of many lncRNAs (fold change <− 2.0), which was not observed in cells with P14AS overexpression alone. This implies that AUF1 may be a dominant factor in regulating P14AS functions.Consistent with the reported functions of AUF1 in degrading RNAs, AUF1 knockdown by siRNA could upregulate the expression of many lncRNAs in HCT116 cells in our RNA-seq analysis. However, in cells with ANRIL expression level was positively associated with the methylation level of the P14 promoter CpG islands in tissues [P14 promoter CpG islands may not be the true ANRIL promoter but the ANRIL gene body CpG islands. We also reported that ANRIL and P16 are coordinately transcribed in CCLE cell lines, and P16 methylation repressed both ANRIL and P16 expression and did not affect P14/P15 expression [P16 methylation repressed P14AS expression in BGC 823 cells  analysis showed that the P16 promoter could play a key role in formation of compact chromatin loops [P16 promoter methylation may lead to focal chromatin condensing and downregulation of these genes. We also found that the 79-nt sequence of P14AS exon 3 was completely overlapped with the 5′-sequence of ANRIL exon 1. To test the probability that P14AS and ANRIL might be spliced from the same primary transcript, we knocked out the promoter-like sequence 2-kb upstream of P14AS exon 1 with CRISPR/Cas9 and found a dramatically decreased P14AS transcription and slightly decreased ANRIL expression in two subclones, providing strong evidence to support the conserved sequence is the P14AS promoter. The fact that KO of the P14AS promoter only weakly decreased ANRIL transcription suggests that the P14AS promoter is likely not the ANRIL promoter or the ANRIL gene could be transcribed using multiple TSSs.It is well-recognized that DNA demethylation of CpG islands around TSS is needed for gene transcription  and that tissues , implyinin loops . P16 proANRIL transcripts could be spliced into diverse mature forms, including linear ANRILs mainly located in the nucleus and circular ANRILs located in the cytoplasm, which imply function difference between linear and circular ANRILs [Unlike mature mRNAs that may contain many exons, approximately 42% of mature lncRNAs contain only two exons . In melar ANRILs . The speANRIL knockdown inhibited the proliferation migration of hepatoma cells [P14AS overexpression or knockout only increased or decreased the proliferation of cell lines HCT116, SW480, and HEK293T, but did not affect the migration of these cells. As we observed in RNA-sequencing data, P14AS could target to both ANRIL and other ncRNAs in AUF1-dependent manner. This may account for the difference of biological functions between ANRIL and P14AS. It is worth studying whether linear or circular ANRIL transcription and degradation are regulated by P14AS.It was reported that ma cells , 34. HowCDKN2A locus hosts the P14AS gene, which might be connected with a true TSS for the ANRIL gene. P14AS is an AUF1-binding lncRNA that upregulates the expression of AUF1 target genes, including ANRIL. P14AS maybe an oncogenic lncRNA involved in the development of colon cancer.The antisense strand of the Additional file 1. Supplementary methodsAdditional file 2 Figure S1. Characterization of P14AS in the CDKN2A/B locus. (A) Sashimi view for transcripts detected by CDKN2A-specific probe-captured RNA (RNACap)-Seq in HEK293T cells containing two wild-type CDKN2A/B alleles and in MCF7 cells with the homogenous P16 deletion. (B) Graphic view of the protein-coding potential for P14AS, CDKN2A/P14, and ANRIL genes (adapted from the UCSC website).Additional file 3 Table S1. List of proteins interacted with P14AS characterized in the HEK293T cells in RNA pull-down mass spectrum analysisAdditional file 4 Figure S2.P14AS affects the expression level of the neighboring genes at 9p21.3. qRT-PCR data are normalized to GAPDH mRNA levels and shown as the means ± SD. (a) The expression levels of P14, P16, and P15 genes in the P14AS vector stably transfected colon cancer cells (HCT116 and SW480), and gastric cancer cells (MGC803) were analyzed by qRT-PCR. (b) Detection of P16/P15/P14 proteins in MGC803 cells in Western blot analyses. (c) A fragment deletion in P14AS exon 1 was detected by PCR (top chart) in HEK293T or HCT116 P14AS-KO clones (KO). The expression changes of P14, P16, and P15 genes in HCT116 and HEK293T cells whose ARE-containing elements in P14AS exon-1 were homogenously deleted in the qRT-PCR analysis (bottom chart). Pooled P14AS ARE-KO-negative subclones were used as a wild-type (WT) control. (d) Detection of the P16, P15, and P14 proteins in HEK293T cells in Western blot analyses.Additional file 5 Figure S3. P14AS expression decreased P16 mRNA-AUF1 binding. (a) AUF1 directly bound to ANRIL and P16 mRNA in HCT116 cells in the AUF1-RIP-PCR. (b) P14AS overexpression decreased ANRIL and P16 mRNA-AUF1 interaction in by the AUF1-RIP-qPCR. (c) An illustration of how the competitive AUF1-P14AS binding protects ANRIL and P16 mRNA from the decay. AUF1 complexes were drawn as dimers based on the reports that AUF1 isoforms  could form functional dimers [l dimers , 26.Additional file 6 Figure S4. Genome-wide analyses of transcriptome by RNA sequencing for HCT116 cells with and without P14AS overexpression and/or AUF1 downregulation. The HCT116 cells with stable P14AS overexpression were transfected with AUF1 siRNAs (siAUF1) for 72 h, and then harvested for RNA sequencing. The number of genes with > 2 fold changes  for different types of RNAs were labeled. Western blot analysis for the determination of AUF1 downregulation by siRNAs was inserted into the top chart. Two samples were sequenced for each group.Additional file 7 Table S2. Function annotations for P14AS-upregulated genes (n = 241) with fold change > 2 in HCT116 cells with the David 6.8: Functional Annotation Tools at the website http://david.ncifcrf.gov/tools.jsp [ools.jsp Additional file 8 Table S3. Function annotations for P14AS-downregulated genes (n = 299) with fold change > 2 in HCT116 cells with the David 6.8: Functional Annotation Tools at the website http://david.ncifcrf.gov/tools.jsp [ools.jsp Additional file 9 Figure S5. Association analyses between the expression levels of AUF1 and ANRIL, P16, or P15 using the publicly available transcriptome databases for human cancer cell lines in the CCLE project. (A) All 1037 cell lines; (B) 224 cell lines without the CDKN2A allele deletion (relative copy number > 0).Additional file 10 Figure S6. Comparison of the levels of P14AS, P16, and P15 expression in colon tissues from cancer and noncancer patients. (a) The expression status of P14AS, P16, and P15 in colon cancer (CC), paired surgical margin (SM), and normal colon biopsy  tissues from noncancer patients by qRT-PCR. (b) The level of P14AS expression in ANRIL-positive and -negative (by RT-PCR) colon CC and SM tissues. (c and d) Comparison of the expression levels of P16 and P15 mRNA (by qRT-PCR) in P14AS-positive and -negative  colon CC and SM tissues. Error bars, S.E.M. *, P < 0.05; **, P < 0.01; N.S. no significance.Additional file 11 Table S4. Comparison of the P14AS expression level (by qRT-PCR) in colon cancer (CC) and surgical margin (SM) tissue samples from patients with different clinicopathological characteristicsAdditional file 12 Figure S7. Comparisons of the levels of AUF1 mRNA (by qRT-PCR) with those of P14AS and ANRIL lncRNA in colon cancer tissues (CCs).Additional file 13 Table S5. Comparison of the ANRIL expression level (by RT-PCR) in colon cancer (CC) and surgical margin (SM) tissue samples from patients with different clinicopathological characteristicsAdditional file 14 Table S6. Comparison of the AUF1 mRNA level (by qRT-PCR) in colon cancer (CC) and surgical margin (SM) tissue samples from patients with different clinicopathological characteristicsAdditional file 15 Table S7. Comparison of P14AS and ANRIL coexpression in colon cancer (CC) and surgical margin (SM) tissue samples from patients with different clinicopathological characteristicsAdditional file 16 Figure S8. Graph of the P14AS gene in the CDKN2A/B locus. (A) CpG islands within the CDKN2A/P14 gene. (B) The transcription and active histone modification status in the chromatin upstream of the P14AS gene in 7 cell lines from ENCODE. (C) Transcription factors binding to various fragments around the P14AS gene from ENCODE. The RNA polymerase II (POLR2A) is highlighted in red lines. (D) The conservation status of various fragments among vertebrates (adapted from the UCSC website).Additional file 17 Figure S9. Repression of P16, P14AS, and ANRIL expression in gastric cancer cells by engineered P16-specific DNA methyltransferase (P16-Dnmt). The gene expression level was detected using qRT-PCR assay. The detailed processes for the construction of P16-Dnmt and transfection experiments are previously described [escribed


This study was designed to explore the roles of P2X3 receptors (P2X3Rs) in the chronic visceral pain induced by combined stress. Here, we showed that P2X3Rs were co-expressed in β2 adrenergic receptor (β2-AR)-positive dorsal root ganglion neurons and that NE significantly enhanced ATP-induced Ca2+ signals. NMD and AMS not only significantly increased the protein expression of P2X3Rs, but also greatly enhanced the ATP-evoked current density, number of action potentials, and intracellular Ca2+ concentration of colon-related DRG neurons. Intrathecal injection of the P2X3R inhibitor A317491 greatly attenuated the visceral pain and the ATP-induced Ca2+ signals in NMD and AMS rats. Furthermore, the β2-AR antagonist butoxamine significantly reversed the expression of P2X3Rs, the ATP-induced current density, and the number of action potentials of DRG neurons. Overall, our data demonstrate that NMD followed by AMS leads to P2X3R activation, which is most likely mediated by upregulation of β2 adrenergic signaling in primary sensory neurons, thus contributing to visceral hypersensitivity.Chronic visceral pain is one of the primary symptoms of patients with irritable bowel syndrome (IBS), which affects up to 15% of the population world-wide. The detailed mechanisms of visceral pain remain largely unclear. Our previous studies have shown that neonatal maternal deprivation (NMD) followed by adult multiple stress (AMS) advances the occurrence of visceral pain, likely due to enhanced norepinephrine (NE)-β Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by intestinal dyskinesia and associated with chronic abdominal pain –3. The c2 adrenergic signaling, while NMD alone does not cause visceral hyperalgesia at the age of 6 weeks[2 adrenergic signaling induces visceral pain remains unknown.More and more evidence has demonstrated that severe adverse environmental factors, especially earlier life stressors may be an inducer that leads to visceral hypersensitivity responses in humans and animals in adulthood –16. Our f 6 weeks. The adrf 6 weeks–20. It hf 6 weeks. However2 adrenergic signaling in visceral hyperalgesia induced by NMD and AMS has not been reported.Adenosine triphosphate (ATP) is an important neurotransmitter in the peripheral and central nervous systems. Its receptors are classified into two subtypes according to the signal transduction mechanisms and molecular structure: the ion channel type P2X receptors (P2XRs) and the metabolic type P2YRs. Recent studies have shown that ATP and its P2XRs may play an important role in nociceptive signaling and pain regulation –24. The 2 adrenergic signaling, which activates P2X3R expression and function, thus contributing to visceral hyperalgesia. To test this hypothesis, we investigated the roles of P2X3Rs in colon-related DRGs using the previously-developed animal model [Therefore, we hypothesize that NMD followed by AMS enhances NE-βal model . This anAdult male Sprague-Dawley rats weighing 150–250 g at the age of 6–7 weeks were selected for the present study. Care and handling of these animals were approved by the Institutional Animal Care and Use Committee of the Soochow University and were strictly in accordance with the guidelines of the International Association for the Study of Pain.NMD rats and age-matched control rats experienced AMS at the age of 6 weeks. The AMS protocol included cold-restraint stress for 45 min, forced swimming stress for 20 min, and water-avoidance stress for 60 min. Rats rested for 1 h between each stressor. These animals were divided into two groups: Control (CON)+AMS and NMD+AMS. The specific methods to establish the NMD+AMS model are described in our previously-published report .Chronic visceral hyperalgesia was measured by recording the minimal distention to evoke an abdominal visceromotor response to colorectal distention (CRD) based on previous studies . All beh2 adrenergic receptor (AR) antagonist, at 5 mg/kg body weight was intraperitoneally injected into the NMD+AMS rats once daily for 7 consecutive days for molecular expression and electrophysiological experiments. In the electrophysiological experiments, ATP (20 μmol/L) or NE (20 μmol/L) was dissolved in extracellular fluid and directly incubated for 5 min with the acutely-dissociated DRG neurons from CON+AMS rats.A317491  dissolved in 0.9% normal saline (NS) was directly injected into the NMD+AMS rats once for behavioral experiments and once daily for 7 consecutive days for molecular expression and electrophysiological experiments. Butoxamine (BUTO), a β2-ARs. The antibodies used were anti-GAPDH , anti-P2X1/2/3R  and anti-β2-AR . Band density was measured using ImageJ software. The P2X1/2/3R and β2-AR expression was normalized to GAPDH.DRGs (T13–L2) from AMS-treated control or NMD rats were dissected out to assess the expression of P2X1/2/3Rs and βp2x3r were: (F) 5′-TTGGGATCATCAACCGAGCC-3′ and (R) 5′-ATGACAAAGACAGAGGTGCCC-3′. The sequences of the primer pairs for gapdh were: (F) 5′-TGGAGTCTACTGGCGTCTT-3′ and (R) 5′-TGTCATATTTCTCGTGGTTCA-3′. The control reaction was performed without a DNA template.Total RNAs were extracted from DRGs (T13–L2) from AMS-treated control or NMD rats with TRIzol . cDNA was synthesized from total RNA using reverse transcription kits  as directed by the supplier’s instructions. The sequences of the primer pairs for 2-AR , gliocyte marker glutamine synthase , neuron marker NeuN , large neuron marker NF200 , small and medium peptidergic neuron marker CGRP , small and medium non-peptidergic neuron marker IB4+ . The secondary antibodies were Alexa Fluor 488  and 555 . Negative controls were without the primary antibodies.Fourteen micrometer (14 μm) frozen sections of DRGs (T13–L2) were used in the immunofluorescence study as described previously , 32. The2PO4, 1.5 CaCl2, 6 MgSO4, 10 glucose, 10 HEPES, pH 7.2, osmotic pressure 305 mOsm. The patch-clamp recording normal external solution (in mmol/L) was: 130 NaCl, 5 KCl, 2 KH2PO4, 2.5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.2, osmotic pressure 295–300 mOsm. The pipette solution (in mmol/L) was: 140 K-gluconate, 10 NaCl, 10 HEPES, 10 glucose, 5 EGTA, 1 CaCl2, pH 7.25, 292 mOsm. The enzyme solution contained collagenase D  and trypsin . The membrane properties were recorded by an HEKA EPC10 patch clamp amplifier . Data were recorded and analyzed using FitMaster (HEKA Electronic GmBH).NMD and control rats exposed to AMS were sacrificed by decapitation (~6 weeks). Detailed procedures for the acute dissociation of DRG neurons and patch clamp recording were as described previously , 31. The2+ mobilization as described previously [2+ peak responses were computed as the difference between the peak value and the baseline value.DRG (T13–L2) neurons were loaded with fura-2 acetoxymethyl ester  with F-127 for 90 min at 37°C. The ratio (R) of fluorescence signal measured at 340 nm, divided by the signal at 380 nm, is proportional to the intracellular Caeviously . The ampt test, the Mann-Whitney test, and other statistical analysis methods as appropriate. P < 0.05 was considered statistically significant.Origin 8 software  was used for statistical analysis. All data are shown as the mean ± SEM, and the error bars represent the SEM. All data were tested for a normal distribution before analysis. Different data were analyzed by the two-sample 2 adrenergic signaling [2-AR in DRG neurons in CON+AMS rats, and immunofluorescence experiments showed that they were  significantly increased after incubation with NE (20 μmol/L) in CON+AMS rats  before NE application. Five minutes after NE application, the ΔR/R was 0.81 ± 0.16 (n = 33). The ATP-induced intracellular Ca2+ mobilization returned to baseline after removal of NE. These data indicated the involvement of adrenergic signaling in the sensitization of P2X3R function.Next, the Caats Fig. C. The ΔR+, but they were less expressed in large neurons labeled with NF200, and small and medium-sized peptidergic neurons labeled with calcitonin gene-related peptide (CGRP). The analysis showed that the percentages of P2X3R-positive cells among CGRP- and IB4+-positive cells were 16.26% and 55.17%, respectively. The percentages of CGRP- and IB4+-positive cells among all P2X3R-positive cells were 18.85% and 59.86%, respectively  were induced by application of ATP (20 μmol/L). The results showed that the current density and number of APs induced by ATP in the NMD+AMS group were significantly higher than those in the CON+AMS group  to NMD+AMS rats. The results showed that the protein expression of P2X3Rs in the BUTO group was markedly lower than that in the NS group of NMD+AMS rats  did not affect the expression of β2-ARs in NMD+AMS rats . Next, we tested their targeting relationship. After intraperitoneal injection of the β2-AR antagonist BUTO into NMD+AMS rats for 7 consecutive days, the protein expression of P2X3Rs was suppressed. This result not only supports our previous finding that BUTO suppresses visceral pain [2-ARs, indicating that P2X3R activation might not be part of the upstream machinery of adrenergic signaling. Together with other findings that application of BUTO suppressed the ATP-induced inward currents and number of action potentials and that NE significantly enhanced the ATP-induced Ca2+ signal in T13–L2 DRGs neurons of CON+AMS rats, it is reasonable to conclude that P2X3R activation is most likely mediated by enhanced adrenergic signaling in the combined stress rat model. This conclusion is consistent with our previous study that adrenergic signaling mediates mechanical hyperalgesia by activating the P2X3Rs of primary sensory neuron in rats with chronic pancreatitis [2-AR signaling might regulate P2X3R protein expression after transcription. Whether there are other mechanisms such as roles of microRNAs involved in this regulation needs to be further explored. It is also important to further investigate the detailed molecular mechanism by which the activation of β2-ARs promotes purinergic receptor activity under combined stress conditions. In addition, the rapid effect of NE-induced Ca2+ signaling might not be mediated by upregulation of P2X3R expression in vitro. The purpose of this experiment was to explore the possible interaction of NE and ATP at the DRG neuronal level. This experiment provides a clue that the NE signaling pathway promotes ATP-induced responses. The detailed mechanism of this phenomenon has not been fully investigated. The possible mechanisms might include but may not be limited to the trafficking of P2X3Rs from the cytosolic compartment to the cell membrane and changes in channel kinetics, including increased open probability.Next, we investigated the mechanisms by which the expression and function of purinergic P2X3Rs were potentiated in NMD+AMS rats. Based on our previous study showing that NE–βral pain , but alsreatitis . Of note2-AR signaling and then activate P2X3R expression and function, thus advancing visceral hyperalgesia. This study helps to elucidate the pathogenesis of visceral hyperalgesia induced by early and adult combined stress. It also suggests that AR inhibitors or P2X3R inhibitors might be alternative drugs to alleviate abdominal pain in IBS patients.Together with our previously report , our fin


Myocardial infarction (MI) is one of most common cardiovascular diseases, and ischemia/reperfusion (I/R) injury is one of the risk factors for severe myocardial injury and dysfunction, even leading to high mortality of myocardial infarction. Liraglutide, a novel glucagon-like peptide 1 (GLP-1) analogue, has been reported to reduce cardiac rupture and infarct size and improve cardiac function in normal and diabetic rodents, however, the mechanisms of liraglutide on cardiomyocytes is not clear. The current research was designed to investigate the hypothesis that liraglutide would protect cardiomyocytes through regulating homer1 expression under hypoxia/reoxygenation (H/R) condition. The results of the present study indicated liraglutide reduced hypoxia-reoxygenation induced cell death and attenuated intracellular calcium overload in H9C2 cardiomyocytes under H/R condition. Moreover, liraglutide significantly increased the Homer1 protein expression, and this protection might be related to Homer1-dependent regulation of endoplasmic reticulum (ER) calcium homeostasis. Taken together, liraglutide protects H9C2 cell against H/R induced cell injury, and this protective effect may inhibit intracellular calcium overload to some extent, through Homer1-dependent regulation of ER calcium homeostasis. Acute myocardial infarction (AMI) is most commonly associated with acute coronary artery occlusion and remains a major cause of high human morbidity and mortality worldwide . Rapid r2+ signaling. Previous studies on Homer mainly focused on the central nervous system (CNS), but for now, more and more researchers began to investigate the role of Homer in cardiovascular disease cyt using Fura-2-AM. Dynamic changes of [Ca2+]cyt, expressed as a percentage of the baseline for up to 12 h following H/R injury was shown in 2+]cyt with 1 h, and then slowly returned to baseline after 12 h. Compared with vehicle cells, liraglutide pretreatment significantly reduced [Ca2+]cyt at 30 min, 1 h, 3 h, and 6 h, suggesting delayed calcium afflux and inhibited Ca2+ overload after H/R treatment.To evaluate the potential role of intracellular CaAfter 4 h after reoxygenation, we investigated the effect of liraglutide on expression of Homer1 in H9C2 cells by RT-qPCR. The results demonstrated that H/R reduced expression of Homer1, but liraglutide reverse the effect. Pre-treatment liraglutide promoted mRNA expression of Homer1 in H9C2 cells against H/R . To furt2+]ER level was significantly increased by LV-H1 transfection as compared on LV-Con transfected H9C2 cells at 10 min and 15 min, suggesting an inhibited ER Ca2+ release and promoted calcium recovery in Homer1 upregulated H9C2 cells following H/R treatment  and control lentivirus (LV-Con) in H9C2 cells. Immunofluorescence staining suggested that exogenous Homer1 was expressed in H9C2 cells after LV-H1 transfection, and the total level of Homer1 was about 4 times higher than that vehicle cells , 5B. As reatment . In addireatment , 6B. As stration .Since the phenomenon of ischemia/reperfusion injury was first described in 1960s, the research and technology on solving this clinicopathological problem has been made great progress in the past few decades . The cliTo determine the protect effect of liraglutide in the myocardia I/R injury, H9C2 cells were pre-treated with liraglutide in different concentrations before H/R treatment. Our results revealed that certain concentration of liraglutide significantly prevented the decrease of cell viability, percentage of death cells and LDH, which treated by H/R. Subsequently, we found that liraglutide not only increased the number of AV-/PI- cells, but also decreased the number of AV+/PI+ cells, indicating that liraglutide has anti-apoptotic activity. Hoechst 33342 staining showed that liraglutide prevented the changes of DNA fragmentation and nuclear chromatin condensation in injured H9C2 cells.2+ overload is the primary stimulators to damage mitochondrial function and induce cardiomyocytes death in H/R condition ER) and cytoplasm ([Ca2+]cyt) as previously described [2+ can produce strong fluorescence under the excitation light of 330-350 nm, while it will lead to fluorescence attenuation under the excitation light of 380 nm. Thus, the ratio of fluorescence of 340 nm and 380 nm can be used to detect intracellular calcium concentration. Using multifunctional luciferase microplate reader , fura-2 were excited at 340 and 380 nm and the emission fluorescence was recorded at 510 nm.Fura-2-AM  was performed to measure intracellular Caescribed . H9C2 ceCoverslips containing attached H9C2 cells were fixed with 4% formaldehyde for 15 min, permeabilized with 0.25% Triton X-100 for 20 min, and closed antibodies with PBS-BSA for 1 hour. Slides were incubated with Homer1 rabbit polyclonal primary antibody  at 4° C in overnight. In addition, cells were cultured with goat resist rabbit secondary antibodies  for 1 h. Then, DAPI stained the nuclei and detected fluorescence.mRNA was reverse-transcribed into cDNA using BeyoRT™II First Strand cDNA Synthesis Kit . Real-time PCR was performed on a Real-time PCR system  using FastFire qPCR PreMix (SYBR Green) . All the assays were independently repeated for 3 times. Homer1 mRNA was amplified through polymerase chain reaction . GAPDH  was used as the internal reference gene for normalization of mRNA levels .Proteins (40 μg) from H9C2 cells were separated by SDS-PAGE gel electrophoresis, which was followed by electroblotting onto PVDF membranes (EMDMillipore). The blot was probed by using respective primary antibodies including Homer1 (1:1000), GAPDH . The bands were analyzed by Hypersensitive ECL chemiluminescence kit .P<0.05). All values are reported as mean ± standard deviation (SD) of the mean.GraphPad7.0 software  was performed to analyze data with independent sample Student’s t-test or one-way analysis of variance analysis of variance test (ANOVA) with post hoc contrasts by Student-Newman-Keuls test (


PIEs expressing different HBGAs  were established and isolation, propagation, differentiation and RV infection conditions were optimized. Differentiated PIEs were infected with human RV (HRV) G1P[8] Wa, porcine RV (PRV) G9P[13], PRV Gottfried G4P[6] or PRV OSU G5P[7] virulent and attenuated strains and virus replication was measured by qRT-PCR. Our results indicated that virulent HRV G1P[8] Wa replicated to the highest titers in A+ PIEs, while a distinct trend was observed for PRV G9P[13] or G5P[7] with highest titers in H+ PIEs. Attenuated Wa and Gottfried strains replicated poorly in PIEs while the replication of attenuated G9P[13] and OSU strains in PIEs was relatively efficient. However, the replication of all 4 attenuate strains was less affected by the PIE HBGA phenotypes. HBGA synthesis inhibitor 2-F-Peracetyl-Fucose (2F) treatment demonstrated that HBGAs are essential for G1P[8] Wa replication; however, they may only serve as a cofactor for PRVs G9P[13] and OSU G5P[7]. Interestingly, contrasting outcomes were observed following sialidase treatment which significantly enhanced G9P[13] replication, but inhibited the growth of G5P[7]. These observations suggest that some additional receptors recognized by G9P[13] become unmasked after removal of terminal SA. Overall, our results confirm that differential HBGAs-RV and SA-RV interactions determine replication efficacy of virulent group A RVs in PIEs. Consequently, targeting individual glycans for development of therapeutics may not yield uniform results for various RV strains.Rotaviruses (RVs) are a leading cause of acute viral gastroenteritis in young children and livestock worldwide. Growing evidence suggests that host cellular glycans, such as histo-blood group antigens (HBGAs) and sialic acids (SA), are recognized by the RV surface protein VP4. However, a mechanistic understanding of these interactions and their effects on RV infection and pathogenesis is lacking. Here, we established a  Cell surface glycans, including histo-blood group antigens (HBGA) and sialic acids (SAs), have been shown to serve as receptors/attachment factors for many pathogens including RVs. However, how those glycans affect RV replication remains largely unknown due the lack of reliable in vitro models. To solve this problem, we established a 3D porcine intestinal enteroid (PIE) model that recapitulates the complex intestinal morphology better than conventional cell lines. By utilizing PIEs expressing different types of HBGAs, we found that several RV strains including Wa G1P[8], OSU G5P[7] and G9P[13] show preference for certain HBGA types. Interestingly, only Wa replication was reduced when HBGAs synthesis was inhibited, while that of OSU and G9P[13] was only marginally affected, which indicates that they may utilize alternative attachment factors for infection. Sialidase treatment strongly inhibited the growth of OSU, while G9P[13] replication was significantly enhanced. These findings suggest that SAs play contrasting roles in the infection of PRV OSU and G9P[13] strains. Overall, our studies demonstrate that PIEs can serve as a model to study pathogen-glycan interactions and suggest that genetically distinct RVs have evolved diverse mechanisms of cell attachment and/or entry. Rotaviruses (RVs) are an important cause of severe diarrheal illness in infants and young animals including pigs . In chilA substantial body of information on RV host cell attachment has been generated RV during the past 20 years. However, the entry mechanism of RV is not yet fully understood. Although the actual cellular receptors of RVs are not confirmed, early studies indicated that the VP4 protein ([P] type) is associated with the initial interaction of RV and sialic acid (SA) ,14, and Subsequent studies suggested that the VP8* of a human RV strain HAL1116 (P[14]) selectively recognizes the type A histo-blood group antigen (HBGAs), highlighting the importance of interactions between RVs and HBGAs ,18. Furtin vitro binding assay indicated that strains of P[4] and P[8] genotypes reacted with H type 1 and Lewis b (Leb) antigens, while strains of P[6] bound to H type 1 only , P[6], P[8] strains which would recognize the type1 precursor and support the interactions of VP8* with ABH antigen, but likely would be unable to recognize Lewis HBGA  Wa strain, porcine G4P[6] Gottfried strain, porcine G5P[7] OSU strain and porcine G9P[13] strains. RV levels were measured by RT-qPCR at 1.5 hours post-inoculation (hpi) and 24hpi. Our results indicated that virulent RVs replicated to higher titers in A+, H+ or A+H+ PIEs in a strain-specific manner . SpecifiTo further confirm the role of HBGAs in RV infection, a fucosyltransferase inhibitor, 2F-Peracetyl-Fucose (2F) ,39,40 waRT-PCR detection of B) Wa, C) G9P[13] and D) OSU infectivity in A+, H+, and H+ PIEs, respectively. Ct values were converted to FFU/ml based on a standard curve which combined CCIF and RT-PCR results. The error bar represents the standard deviation from duplicate samples. All experiments were repeated at least 2–3 times. For P values, **, p<0.01Because we only observed minimal effects of the 2F treatment on OSU and G9P[13] replication, we wanted to further explore the roles of SAs in their replication. OSU has been reported previously to be a sialidase-sensitive strain, while Wa and Gottfried were shown to be sialidase-insensitive strains . Thus, wSince all attenuated strains tested in this study did not demonstrate preference for certain HBGA types, we tested if NA sensitivity of these attenuated strains was also altered compared to their virulent counterparts. The resultant growth curves of attenuated Wa, OSU and G9P[13]  or H+ (for OSU and G9P[13] infection) PIEs were divided into four treatment groups: DMSO-TNC (control group), DMSO-NA , 2F-TNC (2F single treatment group), and 2F-NA . The growth of Wa  did not HBGAs have been reported as potential receptors or attachment factors for RVs –28,42–44Live attenuated oral vaccines like Rotarix  provide good protection to infants and children in developed countries but perform poorly in the developing world –49. The Consistent with our results, an epidemiological study showed that among children admitted to hospitals with RV gastroenteritis , those secretor+ with A or AB blood group types were significantly more prone to acute gastroenteritis caused by the prevalent P[8] RV strains . AlthougOur sialidase treatment results confirmed previous observations demonstrating that replication of sialidase-sensitive strains, like G5P[7] OSU, was significantly inhibited by sialidase treatment, while sialidase treatment showed no inhibition or only a slight increase in the growth of sialidase-insensitive strains like G1P[8] Wa ,64. SurpCollectively, we have successfully established PIEs expressing major porcine HBGA types providing, a physiologically functional in vitro system to comprehensively evaluate RV-host glycan interactions. Our RV infection results strongly support the original hypothesis that replication of virulent RVs is significantly affected by HBGA types expressed on PIEs and this HBGA selectivity is strain dependent. Further, this study demonstrate that the efficacy of live attenuated RV vaccines may not be affected by variable host HBGA phenotypes, because attenuation via cell culture adaptation may reduce RV HBGA selectivity. Furthermore, we concluded that removal of terminal SAs may not only inhibit (as was previously demonstrated), but can also greatly enhance attachment and/or replication of some RV strains. Finally, this research broadens our knowledge on SA-HBGA-RV interactions and provides novel insights into RV evolutionary mechanisms potentially critical for its epidemiology, prevalence, zoonotic transmission, and development of preventative and interventional strategies.All of the pigs were derived, maintained, and euthanized as described in the protocol #2010A00000088 (approved by OSU IACUC) in accordance with guidelines of public health service policy on humane care and use of lab animal and USDA animal awareness act guideline for animal care and use of lab animal . The pigs were euthanized using Telazol-Ketamine-Xylazine intra-muscular injection for anesthesia which was followed by intracardiac injection of pentobarbital.Pairs of virulent and cell-culture adapted (attenuated) human/animal group A RVs used: human G1P[8] Wa strain, porcine G4P[6] Gottfried strain, porcine G5P[7] OSU strain, and G9P[13] RV0084 strain. The virulent strains were serially passaged in gnotobiotic (Gn) pigs for 9 (Gottfried), 12 (OSU), 22 (Wa) and 5 (G9P[13]) passages as described , 65, andMedia used for PIE establishment, maintenance and differentiation were formulated as follows: complete medium without growth factors [CMGF(-) medium] consisted of advanced DMEM/F12 medium (Invitrogen) supplemented with 100 U/ml penicillin-streptomycin (Invitrogen), 10mM HEPES buffer (Invitrogen), and 1X GlutaMAX (Invitrogen) ,33. CompCrypt cells were extracted from the small intestine (ileum) as described by Sato et al. with some modification . BrieflyPIEs were washed using 1X phosphate buffered saline (PBS) at room temperature (RT) after removing CCCM and then fixed with 1ml of 2% paraformaldehyde (PFA) 0.1% glutaraldehyde (GA) in 1X PBS for 30 minutes, RT. Fixed PIEs were washed with 1X PBS 3 times for 10min. After washing, Matrigel domes were collected using a scoop and placed in a 50 ml conical tube containing 20% sucrose in 1X PBS, and the tube was left at 4°C until the domes fall to the bottom of the tube (overnight or longer as needed). Once the domes were in the bottom of the tube, sucrose solution was removed, and the domes were placed in a mold followed by adding Tissue-Tek O.C.T. compound (Sakura) for embedding. Molds were snap- frozen and stored at -80°C until use.For sectioning, a cryotome (LEICA CM 1510S) was used and PIE blocks were cut in approximately 5 or 8 um thick and heat-mediated antigen retrieval was performed on PIE sections using 1X reveal decloaker (BIOCARE). Slides were washed using 1X PBS and treated with 0.15% Triton to increase the permeability of the cell membrane. Then slides were washed again using 1X PBS and blocking was performed using 3% Bovine Serum Albumin (BSA). The following primary antibodies were diluted in 3% BSA: mouse BG-2 mAb , mouse BG-4 mAb , rabbit SOX9 . The slides were incubated with the diluted primary antibodies at 4°C, overnight, and washed using 1x PBS. Then the following secondary antibodies diluted in 3% BSA were added to the respective slides: Alexa Fluor 488 Goat anti-mouse IgG3 , Alexa Fluor 488 Goat anti-rabbit IgG . All secondary antibodies were incubated with slides at RT temperature for 2h. DAPI  was used for nuclear staining and Vector TrueVIEW Autofluorescence Quenching Kit was used to reduce background fluorescence. Slides were sealed by adding mounting medium and pictures were using Keyence BZ-810 microscope.To achieve the correct multiplicity of infection (MOI), 1ml of accutase cell dissociation solution (BD Biosciences) was added to each well and incubated at 37°C for 30min. The cell number was counted by Cellometer Auto T4 (Nexcelom Bioscience). The MOI was calculated as the amount of input virus/total number of cells in samples of PIEs. For RVs infection, PIEs were differentiated in DM for at least 4 days. GCDR was used to remove matrigel and an apical exposure to RV was performed using 200μl pipette as previously described . The RV Arthrobacter ureafaciens (Roche) for 1h at 37°C before inoculation. TNC buffer  were used to dilute sialidase and serve as a control for sialidase treatment. Following 2F or sialidase treatment, PIEs were infected by RVs as described above.2F-Peracetyl-Fucose is a cell-permeable fluorinated fucose derivative that acts as an inhibitor of fucosyltransferases following its uptake and metabolic transformation into a GDP-fucose mimetic,39,40. F-ΔΔCT method as previously described [A lysis buffer containing 0.5mg/ml protease K (Amresco), 50 mM KCl (Sigma), 10 mM Tris-Cl pH 8.0 (Invitrogen), 2.5 mM MgCl2 (Sigma), 0.45% IGEPAL (Sigma), 0.45% Tween-20 (Promega) and DEPC-treated water (Promega) was used for total RNA extraction. Briefly, 200μl of lysis buffer was added in each well in 96-well plate that contains PIEs and was incubated for 30min at 37°C. Then 100μl of the PIEs-lysis buffer mixture was transferred to a 96-well PCR plate followed by incubating in a PCR cycler and using the following parameters: 30min at 65°C, 2min at 98°C and cool down to 4°C. The RNA concentration was tested by Nanodrop and adjusted to the same level. Power SYBR Green RNA-to-CT 1-Step Kit (Thermo Fisher) was applied for Quantitative reverse transcription-PCR (qRT-PCR) according to the manufacturer’s protocol. qRT-CR to measure RV RNA levels was conducted as previously described ,67. For escribed . qRT-PCRescribed . Due to escribed .GraphPad Prism v 5.0  were used for data analysis. Comparison of the quantified virus replication data between different treatments was done using Student t-test unless specified. Differences were considered statistically significant when p ≤ 0.05.S1 FigArthrobacter ureafaciens or TNC buffer (TNC-CT) for 1h at 37°C before inoculation. Then, RVs with 3000 FFU were inoculate with MA104 cells for 1.5h at 370C. Plates were harvest at 1.5hpi, 24hpi, 48hpi, 72hpi and the virus growth were measured by RT-PCR.Growth curve of virulent Gottfried strain after infection of sialidase treated MA104 cells. MA104 cells in a 96 well plate were pre-treated with 10mU sialidase  from (TIF)Click here for additional data file.S1 TableThis table shows the genotyping and phenotyping results of the established PIEs. EXPX indicates experiment and pig numbers, respectively. E3P12, E4P5 and E4P13 were used in this study; however, the results were reproducible in PIEs from different pigs expressing the same HBGAs.(DOCX)Click here for additional data file.


U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3ʹ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease. This study reveals a novel, to our knowledge, connection between the splice factor U2AF1 mutation responsible for myelodysplastic syndrome and ribosome biogenesis. Silencing the expression of the ribosome biogenesis factor NPM1 alters the U2AF1-S34F mutant cell proteome, down-regulating ribosome biogenesis and cell cycle progression factors. U2 Small Nuclear RNA Auxiliary Factor 1  forms a heterodimeric complex with U2AF2 U2AF65) that is primarily responsible for 3ʹ splice site selection 5 that is. Using RRPS19, RPS26, RPS17, RPL5, and RPL11 or assembly chaperones such as TSR2 ribosome maturation factor (TSR2) result in “ribosomopathies,” which preferentially lead to defects in myeloid differentiation and present as anemias such as Diamond-Blackfan Anemia and Shwachman-Diamond syndrome NPM1) .This profile can be further normalized by a weighting function that accounts for the nonlinear relationship between sedimentation coefficient, position in the sucrose gradient, and molecular mass: S is approximately x Svedrup . Here, wipoly(xi). This number, generated for each transcript in each of the 3 cell lines, is the basis for clustering in The final numeric value, reflecting translation efficiency, is ∑w2 and w3, and the polysome fractions are w5, w6, w7, and w8.Alternatively, a simpler measure of translational efficiency is reported , consistAverage polysome profiles were generated from quintiles consisting of 35 genes. Error bars are generated with bootstrapping over 30 random replicates.GSEA analysis was done with GSEA v. 4.0.3 from The Broad Institute using the “GSEA on a pre-ranked gene list” option . Gene seluciferase mRNA levels, RNA was isolated by Trizol (Thermo Scientific), treated with DNaseI and reverse transcribed using Superscript III (Thermo Scientific) according to the manufacturer’s instructions using random hexamers. qPCR was done using iTaq SYBR Green master mix  and run on a CFX96 PCR detection system (Bio-Rad). Luciferase mRNA levels were normalized to tubulin mRNA. Primer sequences for luciferase are TAACGCGGCCTCTTCTTATTT and GATTTGCCTGATTTGCCCATAC. Primer sequences for Tubulin mRNA are CGATATTGAGCGTCCAACCTAT and TTCAGGGCTCCATCAAATCTC.Luciferase plasmids were introduced by lentiviral transduction. Equal number of cells were plated in a 96-well dish. After 24 hours, cells were treated with the Dual-Luciferase Reporter Assay System as per manufacturer’s instructions (Promega). Luciferase activity was measured by Victor plate reader . To quantify Protein biosynthesis was assessed by AHA pulse labeling as previously described  with the6 and max IT set at 50 ms over a mass range of 200–1,800 m/z, followed by MS/MS analysis at a resolution of 45,000 with an ion accumulation target set at 1 × 105, max IT set at 120 ms and first fixed mass set at 105 m/z. MS2 precursor isolation width was setup at 0.7 m/z, normalized collision energy was 29, and charge state 1 and unassigned charge states were excluded.Quantitative MS was done according to the NCI MS core standard protocol. Briefly, cells were lysed, and total protein was quantified using bicinchoninic acid (BCA) reagent (Thermo Scientific). The samples were reduced using DTT (Thermo Scientific), alkylated with iodoacetamide (Thermo Fisher Scientific), and trypsinized (Promega) overnight. TMT tags were conjugated to the peptides and cleaned up using peptide desalting columns (Thermo Scientific). The first dimensional separation of the peptides was performed using a Waters Acquity UPLC system coupled with a fluorescence detector . The dried peptide fractions were reconstituted in 0.1% TFA and subjected to nanoflow liquid chromatography  coupled to high-resolution tandem MS . Peptides were separated using a second-dimension low pH gradient using a 2%–40% ACN over 120 minutes in mobile phase, containing 0.1% formic acid at 300 nl/min flow rate. MS scans were performed in the Orbitrap analyzer at a resolution of 120,000 with an ion accumulation target set at 3 × 10Acquired MS/MS spectra were searched against a human uniprot protein database along with a contaminant protein database, using an SEQUEST and percolator validator algorithms in the Proteome Discoverer 2.2 software . The precursor ion tolerance was set at 10 ppm, and the fragment ions tolerance was set at 0.02 Da along with methionine oxidation included as dynamic modification and TMT6 plex (229.163 Da) set as a static modification of lysine and the N-termini of the peptide.Slides were imaged using a custom epifluorescence microscope equipped with a Plan-Apochromatic 40× (NA 1.4) objective , illuminated with a LED light source  and the emitted fluorescence collected with a CMOS camera . The microscope was controlled using μManager . A custoCells were lysed in 1× RIPA buffer, protein lysate quantified using the BCA kit (Thermo Scientific), boiled in SDS, and run on SDS-PAGE. Proteins were transferred onto nitrocellulose membrane, blocked in 5% milk, and probed with the following antibodies: NPM1 , IPO7 , p53 , CCNA2 , RBM10 , puromycin , tubulin , and actin . For SUnSET, cells were treated with 10 μg/mL puromycin for 1 hour before lysis. The CHX-puromycin sample was treated first with 100 μg/mL CHX for 1 hour. Subsequently, fresh medium containing 10 μg/mL puromycin and 100 μg/mL CHX was added to the cells for another 1 hour before cells were lysed.To assess changes in cell viability, cells were counted using an automated cell counter (Bio-Rad), and an equal number of cells were plated in a 96-well plate. On the sixth day of the siRNA experiment, fresh medium was added that contained 1:10 dilution of the WST-1 reagent (Sigma-Aldrich). Cells were incubated for different time points before the absorbance was measured at 450 nm using a plate reader (Bio-Rad).Apoptotic induction was examined by checking for cleaved Caspase-3 levels using a PE-conjugated anti-Caspase-3 antibody per the manufacturer’s instructions (BD Biosciences). PE levels were examined using an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FCS Express .https://github.com/scappell/Cell_tracking.Cell cycle progression was examined using the Click-it EdU Alexa Fluor 647 kit (Thermo Scientific). Briefly, cells were plated on cover slips. After 24 hours, cells were pulsed in EdU for 10 minutes and then fixed in 4% paraformaldehyde in PBS, permeabilized in 0.1% TritonX in PBS. Cells were then washed twice in 2% BSA in PBS. The EdU was reacted with azide-conjugated fluorophore in the presence of copper sulfate. The click chemistry reaction was allowed to proceed for 1 hour. Cells were washed twice in 2% BSA in PBS. DNA was labeled using Hoechst at a dilution of 1:10,000 mounted on Prolong Gold mounting solution (Thermo Scientific). The fraction of cells in each stage of the cell cycle was quantified using a previously published MATLAB  analysis pipeline  and is awww.geneontology.org) [The genes that were down-regulated at an FC of 1.5 in wt/S34F cells depleted of NPM1 in comparison to wt/wt treated with control siRNA were uploaded into the Panther Database (ogy.org) . The Pan32P-labeled probe overnight and imaged using a Typhoon phosphoimager . To probe for the ITS2, an oligonucleotide was 5ʹ-labeled using PNK enzyme . The ITS2 probe was previously reported [GAGGGAGGAACCCGGACCGCAGGCGGCGGCCACGGGAACTCGGCCCGAGCCGGCTCTCTC. The 5ʹ ETS probe sequence was previously reported [GGCGAGCGACCGGCAAGGCGGAGGTCGACCCACGCCACACGTCGCACGAACGCCTGTC. The pET30-2-GAPDH plasmid,a gift from David Sabatini , was used to generate the GAPDH probe[GAPDH mRNA band intensity.Equal numbers of cells were harvested for all conditions using the Luna Automated Cell Counter (Logos Biosystems). Total RNA was extracted using Trizol (Thermo Scientific) according to the manufacturer’s instructions. The RNA was separated on a denaturing gel and transferred to nylon membrane  overnight as previously described . The memreported , and itsreported , and itsPDH probe, and genPDH probe and normImmortalized myeloid cells were treated with 10 μM OPP  for 1 hour. Cells were fixed and permeabilized. Click chemistry was employed to conjugate a fluorophore to the OPP-containing polypeptides according to the manufacturer’s instructions (Thermo Scientific). Cells were washed 5 times in PBS and analyzed on the LSRII flow cytometer (BD Biosciences). Data analysis was performed using FCS Express (De Novo Software). For CHX-OPP samples, cells were treated first with 100 μg/mL CHX for 1 hour. Subsequently, fresh medium containing 10 μM OPP and 100 μg/mL CHX for another 1 hour before cells were fixed.www.cbioportal.org) as of October 4th, 2019. All adult AML and MDS patient studies were selected. These include “Acute myeloid leukemia or myelodysplastic syndromes ” [Cancer patient data were retrieved from cBioPortal ,48 ” , “Acute , 2016)” , “Acute , 2016)” , and “MyS1 FigX and Y axis show PC 1 and PC 2, which explain 78.3% and 21.7% of the total variance, respectively. The underlying data are in (A) Heat map and hierarchical clustering of the polysome/monosome estimator applied to RNA-seq from polysome fractions. The genes are the 175 translation initiation genes present in all 8 fractions of each sample  cells . Rows are centered; no scaling is applied to rows. Rows are clustered using Euclidean distance and Ward linkage. Columns are clustered using correlation distance and average linkage. 175 rows, 3 columns. (B) Principal component analysis of polysome/monosome estimator applied to RNA-seq from polysome fractions. No scaling is applied to rows; SVD with imputation is used to calculate principal components. (TIF)Click here for additional data file.S2 Fig(A) Polysome profile for the top quintile mRNAs. (B) Polysome profile for the middle quintile mRNAs. (C) Polysome profile for the bottom quintile mRNAs. The underlying data are in (TIF)Click here for additional data file.S3 Fig(A) The top 4 GO categories show similar FDR and FWER and contain many of the same genes. The underlying data are in (TIF)Click here for additional data file.S4 Fig(A) Representative western blot of puromycin-labeled nascent polypeptides. (B) Quantification of the signal intensity. Each bar represents the average and standard error of 3 independent experiments. The underlying data are in (TIF)Click here for additional data file.S5 FigNPM1-luciferase mRNA levels were normalized to TUBA1A mRNA. Each bar represents the average and standard error of 3 independent experiments. The underlying data are in NPM1, Nucleophosmin 1; RT-PCR, reverse transcription–quantitative PCR; S34F, serine-34 to phenylalanine substitution; TUBA1A, tubulin alpha 1a; wt, wild-type.The (TIF)Click here for additional data file.S6 Fig(A) Gating strategy from a representative cleaved Caspase-3 experiment. (B) Gating strategy from a representative EdU labeling experiment for NPM1 siRNA. (C) Gating strategy from a representative EdU labeling experiment for IPO7 siRNA. DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-2ʹ-deoxyuridine; IPO7, Importin 7; NPM1, Nucleophosmin 1; PE-A, Phycoerythrin-Area; siRNA, small interfering RNA; SSC-A, side scatter-Area(TIF)Click here for additional data file.S7 Figp < 0.05 using a paired 2-sided t test. (B) Quantification of the ITS2 northern blot showing wt/wt and wt/S34F cells that overexpress an empty vector or the NPM1r-2A-GFP reporter and were treated with control or NPM1 siRNA. Each bar represents the average and standard error of 3 independent experiments. *p < 0.05 using a paired 2-sided t test. (C) Quantification of the 5ʹ ETS northern blot showing wt/wt and wt/S34F cells that overexpress an empty vector or the NPM1r-2A-GFP reporter and were treated with control or NPM1 siRNA. Each bar represents the average and standard error of 3 independent experiments. *p < 0.05 using a paired 2-sided t test. The underlying data are in (A) Quantification of the ITS2 northern blot showing wt/wt and wt/S34F cells treated with control or NPM1 siRNA. Each bar represents the average and standard error of 3 independent experiments. *(TIF)Click here for additional data file.S1 TableS34F, serine-34 to phenylalanine substitution; wt wild-type.(XLSX)Click here for additional data file.S2 TableGeometric means of the quantifications from both assays is shown. MS, mass spectrometry(XLSX)Click here for additional data file.S3 TableThree replicates were run for each sample. Geometric means of the replicates are shown. MS, mass spectrometry; NPM1, Nucleophosmin 1; siRNA, small interfering RNA; S34F, serine-34 to phenylalanine substitution; wt, wild-type.(XLSX)Click here for additional data file.S4 TableAML, Acute Myeloid Leukemia; MDS, Myelodysplastic Syndrome.(XLSX)Click here for additional data file.S5 TableS34F, serine-34 to phenylalanine substitution; wt, wild-type.(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.