In vitro, treatment with FS-115 efficiently blocked p70S6K activity in breast cancer cell lines and impaired colony formation and anchorage independent growth. Pharmacokinetic profiling showed that FS-115 exhibited high oral bioavailability, optimal plasma distribution and high brain penetrance. In nude mice, FS-115 strongly suppressed tumor take-rate and primary tumor growth. Oral dosing with FS-115 in a peri-operative schedule was effective in decreasing local recurrence of breast cancer and a long-term treatment schedule was well tolerated and efficiently suppressed distant metastasis formation. Altogether, we propose that FS-115 might be a good candidate for the treatment of breast cancer patients at high risk to relapse.Breast cancer is a frequent and treatable disease. However, when recurrent, breast cancer often becomes refractory to therapy and progresses into metastatic forms that are typically incurable. Thus, understanding and targeting the critical pathways underlying breast cancer recurrence is urgently needed to eradicate primary disease and achieve better prognosis. Recently, we have demonstrated that the ribosomal protein p70S6K is activated in residual breast cancer cells as a result of post-surgical inflammation and that interfering with its activity in the peri-operative setting strongly suppresses recurrence in a mouse model. In order to develop clinically-exploitable treatments targeting p70S6K, we have tested a newly generated compound, called FS-115. FS-115 potently inhibited p70S6K1 (ICOur results confirm that inhibition of p70S6K represents a valuable opportunity for restraining loco-regional relapse and metastasis in breast cancer and identify in FS-115 a promising candidate-inhibitor to move from preclinical to clinical treatments. Following growth factor or hormones stimulation, or nutrient inputs, the PI3K/mTOR pathway is activated, crucially integrating extra- and intra-cellular signals thereby finely regulating cell survival, growth and metabolism, inducing numerous anabolic processes, including protein and lipid synthesis  . After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 μL of a 3% phosphoric acid solution. 10 μL of the reaction mixture is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting. The concentration of ATP used was 15 μM.FS-115 ability to block p70S6K1 kinase activity was tested using radiometric ATP-competitive kinase assay (Eurofins Millipore KinaseProfiler™ Service Assay). The broader kinase selectivity profile was determined by counter-screening FS-115 against a panel of 268 kinases, excluding p70S6K1, using the Eurofins Millipore KinaseProfiler™ Service. Full details are available at TM System (Promega) protocol and using Genemapper ID Ver 3.2.1, to identify DNA STR profiles. MDA-MB-231-luciferase-expressing cells (MDA-MB-231-luc) derived from MDA-MB-231 cells originally purchased from ATCC. For in vivo experiments, these cells were stably transfected with pGL4.51 (Promega) using Lipofectamine 2000 (Life Technologies), by Axis Bioservices .MDA-MB-231 , MDA-MB-453 , MCF-7  mammary carcinoma cell lines were obtained from ATCC (LGC Standards) and grown in Dulbecco modified Eagle medium  supplemented with 10% fetal bovine serum . All cell lines were authenticated by BMR Genomics srl , according to Cell ID MDA-MB-231, MDA-MB-453, MCF-7 mammary carcinoma cell lines were serum starved in DMEM containing 0.1% bovine serum albumin  and then stimulated with 5% WF for 20 minutes. Where indicated, cells were also pre-treated for 30 minutes with PF-4708671  or FS115 (10 or 50 μM).3VO4 (SIGMA), 10 mM NaF (SIGMA) and 1 mM DTT (SIGMA).To extract total proteins cells were scraped on ice using cold RIPA lysis buffer  plus a protease inhibitor cocktail  and supplemented with 1 mM NaFor immunoblotting analysis, proteins were separated in 4-20% SDS-PAGE  and transferred to nitrocellulose membranes . Membranes were blocked with 5% dried milk in TBS-0.1% Tween20 or in Odyssey Blocking Buffer  and incubated at 4°C overnight with primary antibodies. Then, membranes were incubated 1 hour at RT with IR-conjugated  secondary antibodies for infrared detection .Primary antibodies directed against: AKT (sc-1618), ERK1 (sc-94), p70S6K1 (sc-8418), Src (sc-19), STAT3 (sc-482) and vinculin (sc-7694) were purchased from Santa Cruz; pT202/204 ERK1/2 (#9101), pS473 AKT (#4060), pT389 p70S6K1 (#9234), S6 (#2217), pS235/236 S6 (#4858), pS240/244 S6 (#5364), pS2448 mTOR (#2971), mTOR (#2972), pT37/46 4EBP1 (#2855), 4EBP1 (#9644), pY416 Src (#2101), pY705 STAT3 (#9131), pS3 Cofilin (#3311) were purchased from Cell Signaling; Cofilin (#612145), FAK (#610088) were purchased from BD Biosciences; pY397 FAK (#44624G) was purchased from ThermoFisher Scientific; Actin (#A5060) was purchased from SIGMA.Wound fluid collection. Drainage wound fluids (WF) were collected over the 24hrs after surgery from unselected patients undergone breast-conserving surgery, as described previously . The assFor colony assay, MDA-MB-231 untreated or pre-treated for 24 hours with PF-4708671  or FS-115  were trypsinized, counted and seeded at density 1000 cells/100 mm dish and incubated in complete growth medium in the presence of the inhibitors or left untreated. Two weeks later plates were stained with crystal violet and colonies were counted.4) were re-suspended in 2 ml top agar medium  in the presence of PF-4708671  or FS-115 (10 or 20μM), and quickly overlaid on a previously gelified 0.6% bottom agar medium . The experiments were performed in six-well tissue culture plates, in triplicate. Fresh medium was added to the wells twice a week as a feeder layer. After three weeks, the number of colonies was counted at 4X magnification.To evaluate the anchorage-independent cell growth, MDA-MB-231 cells (1.5×10Pharmacokinetics (PK) studies were conducted by Nikem (IT) and Pharmidex (UK), in male CD-1 mice, looking at profiles from both intravenous (i.v.) and oral (p.o.) routes of administration. FS-115 was formulated using a solution comprised of 3% DMSO, 0.1M aqueous HCl (stoichiometric amount) and a 20% w/v aqueous solution of β-cyclodextrin (dose volume 5 ml/kg). Following a single dose of FS-115 at 10 mg/kg p.o. and 2 mg/kg i.v., plasma was sampled at 8 time points  over 24 hours, followed by protein precipitation using methanol and analysis by LC-MS/MS. Brain-penetrance of FS-115 was determined by a separate discrete oral PK experiment in male CD-1 mice. Following a single dose of FS-115 at 25mg/kg p.o., plasma and brain was sampled at 3 time points , followed by protein precipitation using acetonitrile and analysis by LC-MS/MS.nu) implanted with MDA-MB-231 cells into the thoracic mammary fat pad (1×106 in Matrigel). The mice were randomly assigned to treatment groups of either vehicle QD x 3 PO or FS-115 125 mg/kg BID x 3 PO  when their tumors reached 50-100 mm3. Vehicle used for both treatment groups was 5% DMSO, 95% aqueous hydroxypropyl-β-cyclodextrin (20% w/v). Twelve hours after the final dose animals were sacrificed and the tumor resected. Tumor tissue was homogenized in three volumes Pathscan Sandwich ELISA lysis buffer . Protein concentration was measured using a commercial BCA kit (Sigma) and ELISAs for phospho-S6 240/244, total-S6, phospho-AKT 473 and total-AKT carried out according to manufacturer's instructions .Pharmacodynamics (PD) was conducted by Axis Bioservices in 6 female athymic nude mice . After the appearance of palpable primary tumors, animals were orally treated with FS-115 (125mg/kg) twice a day (BID) in a peri-surgical treatment schedule . Mice were sacrificed at the end point of the experiment (8 weeks from surgery) unless they displayed suffering behavior, in adherence with ethical guidelines of CRO of Aviano Institutional Organism For Animal Wellbeing (OPBA). Tumors, recurrences, lymphnodes and other organs were collected and stored for subsequent analyses.For local recurrence studies, primary tumors were established by injection of 2×10nu), aged 6-8 weeks.Tumor initiation, tumor growth and metastasis studies were conducted by Axis Bioservices  in female athymic nude mice  and then imaged using a Xenogen IVIS machine. Evidence of light only in the cardiac area is indicative of a failed implantation and so any such animals were discarded from the study. FS-115 (or vehicle) was dosed orally via QD (at 250mg/kg) or BID (at 125mg/kg) regimen, with dosing commencing the day prior to inoculation (day -1) and ending 70 days after inoculation. Animals were imaged on 2 occasions during the study, on day 38 from commencement of dosing (mid-point) and on day 70 (end of study).For metastasis study, animals were initially assigned to treatment groups. On day 0, 1×10via QD (at 250mg/kg) or BID (at 125mg/kg) regimen, with dosing commencing the day prior to inoculation (day -1) and ending 21 or 30 days after cell implantation. MDA-MB-231 cells (1×106 in matrigel) were implanted into the second mammary fat pad, on day 0. Time to appearance and tumor volume were estimated respectively by palpation and caliper measurements.For tumor initiation and growth studies, female nude mice were randomly assigned to treatment groups: FS-115 (or vehicle) was dosed orally in vivo experiments, dosing volume of FS-115 was 10 ml/kg and formulation was 5% DMSO, 95% aqueous hydroxypropyl-β-cyclodextrin (20% w/v). Dosing formulation was made up fresh daily. Bodyweight and general clinical signs  were annotated daily and used as a measure of drug tolerance.In all The IC50 graphs were plotted using XLFit version 5.3 (ID Business Solutions). Sigmoidal dose-response (variable slope) curves are fitted using non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Data were examined using the two-tailed Student t test or unpaired two-tailed Mann-Whitney U test. Differences were considered significant at p < 0.05. The computer software PRISM  was used to make graphs and all statistical analyses, unless otherwise specified.


Caenorhabditis elegans senses gentle touch along the body via six touch receptor neurons. Although genetic screens and microarray analyses have identified several genes needed for touch sensitivity, these methods miss pleiotropic genes that are essential for the viability, movement, or fertility of the animals. We used neuronally enhanced feeding RNA interference to screen genes that cause lethality or paralysis when mutated, and we identified 61 such genes affecting touch sensitivity, including five positive controls. We confirmed 18 genes by using available alleles, and further studied one of them, tag-170, now renamed txdc-9. txdc-9 preferentially affects anterior touch response but is needed for tubulin acetylation and microtubule formation in both the anterior and posterior touch receptor neurons. Our results indicate that neuronally enhanced feeding RNA interference screens complement traditional mutageneses by identifying additional nonviable genes needed for specific neuronal functions. Caenorhabditis elegans genome contains 20,517 genes ] (CB47 [unc-11(e47) I], GS2526 , RB1887 [tom-1(ok2437) I], VC206 (fzy-1&cyp-44A1(ok312) unc-4(e120)/mIn1 [mIs14dpy-10(e128)] II), VC3455 (hT2 [bli-4(e937) let-?(q782) qIs48] I+/; kin-18(ok395)/hT2 III), VC556 {hT2 [bli-4(e937) let-?(q782) qIs48] I+/; txdc-9vps-16(ok776)/hT2 III} (note: this paper renames tag-170 as txdc-9), SSM42 , CB408 [unc-43(e408) IV], KY46 [cab-1(tg46) X], MT7553 , VC724 (mog-5(ok1101)/mIn1 [mIs14dpy-10(e128)] II), PK172 (ptc-1(ok122) unc-4(e120)/mnC1dpy-10(e128) unc-52(e444) II), MT8626 [goa-1(n3055) I], VC1148 (vha-5(ok1588)/nT1 [qIs51] IV; nT1 V+/), VC927 (hT2 [bli-4(e937) let-?(q782) qIs48] I+/; pnk-1(ok1435) I/hT2 III), VC2226 (cgt-3(ok2877)/mIn1 [mIs14dpy-10(e128)] II), RB1278 (let-502(ok1283)/hT2 [bli-4(e937) let-?(q782) qIs48] I; hT2 III+/}, and VC1400 (cdk-1(ok1882)/ hT2 [bli-4(e937) let-?(q782) qIs48] I; hT2 III+/) were from the Caenorhabditis Genetics Center; TU3595 dpy-20(e1282) IV; mec-17puEx924], TU4707 (hT2 [bli-4(e937) let-?(q782) qIs48] I+/; txdc-9vps-16(ok776)/hT2 III; mec-17puEx925) were from laboratory stocks. TU4707 and TU4706 were made by injecting VC556 and GS2526 with the indicated constructs, respectively.mec-3p::txdc-9 and mec-3p::mca-3c were made using the Multisite Gateway Three-Fragment Vector system . The plasmid mec-17p::rfp and the entry clones containing mec-3p and unc-54 3′utr were described by txdc-9 genomic sequence was amplified using the primers GGGGACAAGTTTGTACAAAAAAGCAGGCTTAatggccgctaatattcaacagcagt and GGGGACCACTTTGTACAAGAAAGCTGGGTGctaccaatcctcttcgttatcgtactcc. mca-3c cDNA sequence was amplified using primers GGGGACAAGTTTGTACAAAAAAGCAGGCTTAatgcccgaatatggtgcatcactg and GGGGACCACTTTGTACAAGAAAGCTGGGTGtagattgttcgtctccttgatatttccacg. These fragments were then inserted into pDONR221 using standard Gateway BP reactions to create the entry clones.The rescuing constructs of http://ws200.wormbase.org/) using WormMart  using the following phenotype codes: lethal, adult_lethal, embryonic_lethal, embryonic_terminal_arrest_variable_emb, embryonic_lethal_late_emb, larval_lethal, larval_arrest, late_larval_lethal, late_larval_arrest, paralyzed, one_cell_arrest_early_emb, and embryonic_terminal_arrest_variable_emb. The search was restricted to RNAi experiments described by The list of lethal RNAi was pulled from WormBase WS200 . To reduce false-positive results, we performed multiple independent tests and scored an RNAi construct positive only if it produced touch insensitivity four of five times. Because the false-positive rate must be equal to or less than the total positive rate of 20%, the theoretical upper limit for the false-positive rate for five tests is We tested touch sensitivity as described by MEC-7 and MEC-18, a Zeiss Apochromat 100× 1.4 oil objective for antibody staining against acetylated tubulin and MEC-2, and a Zeiss Neofluor 10× 0.3 objective for green fluorescent protein (GFP) imaging. The images were acquired with a Photometrics Coolsnap HQ2 camera using Zeiss Colibri 2 as the excitation source.Animals were immobilized on 4% agarose pads with 30 mM sodium azide and imaged on a Zeiss Observer Z1 inverted microscope using a Zeiss Apochromat 40× 0.95 air objective for TRN antibody staining against Single-molecule mRNA FISH was performed as previously described , mouse anti-MEC-2 . These genes, which are known to affect touch sensitivity , a gene needed for the transport of dsRNA into cells  affected mechanosensation, we performed neuronally enhanced RNAi in strain sitivity . Twelve P < 0.05 after Bonferroni correction), indicating that our screen had a low false-positive rate. Most of the mutants also showed defective posterior touch response to varying degrees (pnk-1 was not significantly different from wild type after Bonferroni correction).We tested the touch sensitivity of animals carrying loss-of-function alleles for 18 of the 61 genes. These strains either carried partial loss-of-function alleles or null alleles that were maternally rescued and, thus, produced young larvae whose touch sensitivity could be tested. All mutants tested produced anterior touch insensitivity  was viable and caused a partial reduction of touch sensitivity . MEC-18 is expressed exclusively in the TRNs; except for the nucleus, MEC-18 is found throughout the TRNs. Eleven of the strains had grossly normal anterior (ALM) and posterior (PLM) TRNs, demonstrating that these genes are not needed for process outgrowth and that the cells have been generated.To look for morphologic defects in the TRNs, we stained animals carrying mutations in 13 of the 18 genes with an antibody against tom-1 and tag-170 (described below) had defective TRNs. Both the ALM and PLM neurons in tom-1 mutants had longer posterior processes. We do not know whether this phenotype is related to the touch insensitivity seen in these animals, especially because tom-1 is known to be involved in synaptic vesicle exocytosis  arrested at various larval stages , we compared maternally rescued txdc-9 animals from a balanced heterozygous strain (VC556) and their heterozygous siblings. Some of the txdc-9(ok776) showed movement defects or were paralyzed. The maternally rescued txdc-9 animals were insensitive to anterior touch, but retained partial posterior touch sensitivity , but single-molecule mRNA fluorescence in situ hybridization . These results indicate that TXDC-9 is needed for tubulin acetylation in both the ALM and PLM neurons, but it may be more critical for microtubules in the ALM neurons.Consistent with a role of -tubulin  and no aMEC-2 and MEC-18 , suggesting defects in the transport of MEC-2 along the process. Similar defects were seen for both MEC-2 and MEC-18 in the ALM and PLM neurons, suggesting that these changes are unlikely to account for the differential touch sensitivity in txdc-9 mutants. The reduced expression and transport defects in MEC-2 and MEC-18 distribution are consistent with defective microtubule organizations in these cells  or function similarly with other genes that are likely to be essential for viability (such as nars-1 and hars-1). These additional genes were not identified in our screen, perhaps because they act redundantly, the RNAi was ineffective, or the genes were not essential for survival and thus were not screened.We have systematically screened genes that cause nonviable phenotypes when mutated using tissue specific RNAi and have identified 61 genes that potentially affect mechanosensation in the TRNs (including five positive controls), out of 849 genes screened . This ramog-5, was confirmed using a maternally rescued null allele. Additional genes may also be needed for cellular housekeeping but were not discovered in our screen, probably due to redundancy.In addition to genes needed for mechanosensation, we expected that this screen would also uncover genes needed for general cell functions. Indeed, at least 12 of the candidate genes are involved in cellular housekeeping functions, including general transcription factors, RNA polymerase subunits, and tRNA synthetases. One of these genes, A total of 17 other nonhousekeeping genes were confirmed using maternally rescued null alleles or partial loss-of-function alleles. The nonhousekeeping genes, however, do not necessarily function specifically in mechanosensation. Candidate genes in several groups, including protein degradation, calcium signaling, cytoskeleton, mitochondria, and synaptic functions, are likely to affect neuronal functions in general. Some of these genes, nevertheless, may have specific roles in the TRNs.txdc-9, is needed for microtubule function and is conserved from yeast to humans , G proteins (goa-1), and Rho GTPase (let-502), suggesting that these signaling pathways may also affect the function or the development of the TRNs.Genes involved in classical signaling cascades may affect TRN functions more specifically. A previous RNAi screen for genes involved in integrin signaling found that the insulin-like pathway is involved in gain control in the TRNs . Hilliarpxl-1, encoding a Paxillin, likely functions with the other focal adhesion proteins (hmr-1 encodes a classical cadherin that functions independently of the focal adhesion complexes. This result suggests that the cadherin complexes also may contribute to mechanosensation in the TRNs.Genes involved in focal adhesion complexes have been shown previously to be involved in TRN functions . In thisIn summary, our results suggest that screens using neuronally enhanced feeding RNAi complement traditional forward mutagenesis screens in identifying genes needed for neuronal functions. The interpretation of the candidates from such screens, however, requires confirmation and further analysis using null alleles. In addition, mosaic analysis can be used to overcome lethality by testing animals lacking the essential genes specifically in the TRNs.


Colorectal cancer (CRC) is the second leading cause of preventable cancer-related deaths, and Blacks and Latinos are disproportionately affected. While CRC screening reduces cancer disparities, barriers exist at the system, clinician and patient levels. Prevention Care Management (PCM) has increased CRC screening rates in eleven CHCs and four Medicaid Managed Care Organizations (MMCOs).Three consecutive randomized controlled trials (RCTs) tested whether PCM improves CRC screening among low-income, uninsured and Medicaid-insured women aged 50-64 in Community Health Centers (CHCs) and other primary care practices and Health Plans. We present here the spectrum of research from efficacy (PCM1), to effectiveness (PCMT), to dissemination and implementation (PCM2), to scale-up and sustainability (PCM3), and now examine the contribution of patient and community-based organization (CBO) stakeholder engagement to scale-up and sustainability.Health education/outreach staff, initially from one practice-based research network (PBRN) and four MMCOs, and now a second PBRN, three CHCs and two CBOs, provide structured telephone support in English, Spanish or Russian, to identify barriers and facilitators to receiving CRC screening. Women receive educational materials and were followed up using chart/EHR (PCM1/PCM3) and administrative/claims (PCMT/PCM2) data.PCM addressed barriers at patient ; clinician (lack of clinician recommendation); and systems (difficulty making appointments and long waiting times for procedure) levels. CRC screening increased significantly (P < 0.05) in CHCs (OR = 1.60) and Health Plans (OR = 1.44), as compared to Usual Care with no significant heterogeneity . Overall, PCM1/PCM2 effects are stronger for Spanish-speaking women (OR = 1.92/1.81) versus English-speaking women (OR = 1.38/1.13).Implementation Science Conclusions: PCM is robust and transferable, increases CRC screening, and can be disseminated and implemented successfully across a wide range of settings in underserved communities. Effective and sustainable PCM interventions address multi-level barriers, and should be integrated into primary care.New York Prevention Care Management Project (PCM1) NCI 3R01 CA87776, Dartmouth Medical School (09/01/2000-08/31/2004) New York Prevention Care Manager Translation Pilot (PCMT) NCI 3R01 CA87776-04-S1, Dartmouth Medical School (9/01/2004 -8/31/2005) New York Prevention Care Management Project 2 (PCM2) NCI R01 CA119014-01, Dartmouth Medical School (08/01/2006-07/01/2011) New York Prevention Care Management Project 3 (PCM3)/ "Collaborative Care to Reduce Depression and Increase Cancer Screening Among Low-Income Women" PCORI IH-12-11-4522, Albert Einstein College of Medicine/Montefiore Medical Center (08/01/2006-07/01/2011).


The dual-luciferase reporter assay further validates that lncRNA-NUTF2P3-001 and 3′UTR of KRAS mRNA competitively bind with miR-3923. Furthermore, miR-3923 overexpression simulates the inhibiting effects of lncRNA-NUTF2P3-001-siRNA on pancreatic cancer cell, which is rescued by miR-3923 inhibitor. Specifically, the present study further reveals that lncRNA-NUTF2P3-001 is upregulated in pancreatic cancer cells under hypoxia and CoCl2 treatment, which is attributed to the binding of hypoxia-inducible factor-1α (HIF-1α) to hypoxia response elements (HREs) in the upstream of KRAS promoter. Data from pancreatic cancer patients show a positive correlation between lncRNA-NUTF2P3-001 and KRAS, which is associated with advanced tumor stage and worse prognosis. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumor oncogene KRAS and implicate that lncRNA-NUTF2P3-001 and miR-3923 can be applied as novel predictors and therapeutic targets for pancreatic cancer.Recent studies indicate that long non-coding RNAs (lncRNAs) play crucial roles in numerous cancers, while their function in pancreatic cancer is rarely elucidated. The present study identifies a functional lncRNA and its potential role in tumorigenesis of pancreatic cancer. Microarray co-assay for lncRNAs and mRNAs demonstrates that lncRNA-NUTF2P3-001 is remarkably overexpressed in pancreatic cancer and chronic pancreatitis tissues, which positively correlates with KRAS mRNA expression. After downregulating lncRNA-NUTF2P3-001, the proliferation and invasion of pancreatic cancer cell are significantly inhibited both  The long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with more than 200 nucleotides in length , which aSome lncRNAs are identified as competing endogenous RNAs (ceRNAs) which act as molecular sponges for a microRNA through their miRNAs binding sites , thereby derepressing the targets of the respective miRNAs. LncRNA-PTENP1 has been discovered as ceRNAs to protect PTEN from miRNAs-mediated degradation . RecentlRecent studies have demonstrated that the lncRNAs can be regulated under hypoxia in cancer , 27. In vitro and vivo. Meanwhile, the dual-luciferase reporter assay was applied to verify the competitive binding of lncRNA-NUTF2P3-001 and KRAS mRNA with miR-3923. Furthermore, the knock-in and knock-out of miR-3923 were performed to confirm that lncRNA-NUTF2P3-001 upregulates KRAS expression by depriving the inhibition of miR-3923 on KRAS. More importantly, ChIP assay and HREs luciferase reporter assay were further adopted to identify that lncRNA-NUTF2P3-001 is transcriptionally upregulated by HIF-1α in pancreatic cancer cell.In present study, the positive correlation between lncRNA-NUTF2P3-001 and KRAS mRNA was further validated in normal pancreas, chronic pancreatitis and pancreatic cancer. Moreover, gain and loss experiments of lncRNA-NUTF2P3-001 were performed to evaluate its role in the proliferation and invasion of pancreatic cancer cell in both Hierarchical clustering analysis of microarray data showed genetic alterations in the expression of both lncRNAs and mRNAs among pancreatic cancer (PC), chronic pancreatitis (CP) and noncancerous pancreatic (NP) tissues Figure  left. Coin vitro and might play a role via regulating KRAS and its downstream pathways. Despite being statistically significant, differences on some functional assays and expression levels of KRAS signaling elements are moderate might due to the limited efficiency of siRNA-mediated silencing.Since lncRNA-NUTF2P3-001 was upregulated in pancreatic cancer samples, we downregulated lncRNA-NUTF2P3-001 with lncRNA-NUTF2P3-001-siRNA (NUTF2P3-001-siRNA) to further identify its functional roles in pancreatic cell lines PANC-1 and BXPC-3. Three siRNA sequences were designed and the most effective one was chosen to fulfill the following experiment . After dAccording to recent researches, large fraction of the lncRNAs present in mammalian cells is found to localize within cell nucleus and some of them participate in regulation of key nuclear processes. However, in general the efficiency of siRNA-mediated depletion of nuclear-retained RNAs is low due to the fact that the siRNA machinery is located mainly in the cytoplasm of mammalian cells , 31. Henin vitro via regulating KRAS and its downstream pathways on the other hand.After overexpression with pcDNA-NUTF2P3-001, the viability of pancreatic cancer cell was significantly promoted . MoreoveWe further identified the underlying regulative mechanisms of lncRNA-NUTF2P3-001 on KRAS expression. The miRNAs target prediction algorithm suggested that both lncRNA-NUTF2P3-001 and the 3′UTR of KRAS mRNA had potential binding sites for miR-3923 or miR-19b-3p . HoweverTo further reveal the function of miR-3923 in pancreatic cancer, we tested the expression of miR-3923 in pancreatic cancer and chronic pancreatitis tissues by qRT-PCR. Compared with the noncancerous pancreatic tissues, obvious reduced miR-3923 was observed in pancreatic cancer but not chronic pancreatitis specimens . NeverthTo further identify the pivotal role of miR-3923 in crosstalk between lncRNA-NUTF2P3-001 and KRAS mRNA, the biological behaviors of pancreatic cancer cell were observed after miR-3923 mimics transfection. As shown in To further identify the interaction between miR-3923 and lncRNA-NUTF2P3-001 in the regulation of KRAS expression, we transfected miR-3923 inhibitor into PANC-1 cells treated by LV-NUTF2P3-001-siRNA. The data revealed that miR-3923 inhibitor significantly promoted KRAS expression Figure , 4B, viain vivo, the PANC-1 cells transfected with LV-NUTF2P3-001-siRNA, LV-NC-siRNA, LV-miR-3923 and LV-miR-NC were injected subcutaneously into BALB/c athymic nude mice of 3-week-old (n = 5 per group) respectively. Compared with associated NC group, the growth rate  and then enhances target genes expression, including lncRNAs [2, while decreased with the HIF-1α-siRNA. Moreover, the combination and activity of HIF-1α with promoter of lncRNA-NUTF2P3-001 was further confirmed by the ChIP and luciferase reporter assay respectively. Therefore, all of these data intensively suggests that lncRNA-NUTF2P3-001 is transcriptionally regulated by HIF-1α in hypoxia of pancreatic cancer.As a result of hypoxia in pancreatic cancer, hypoxia inducible factor-1 (HIF-1) is significantly overexpressed in pancreatic cancer tissue and cell , 35. HIF lncRNAs , 27. Xue lncRNAs . On the  lncRNAs . Since tIn summary, our work highlights the importance of the complicated miRNAs-lncRNA network in modulating the cellular response to hypoxic microenvironment of pancreatic cancer. Furthermore, our findings demonstrate the crucial role of lncRNA-NUTF2P3-001 mediated translational desuppression in KRAS expression and the therapeutic potential of targeting this pathway in pancreatic cancer.125 seeds as well as choledochojejunostomy and gastroenterostomy, depending on the National Comprehensive Cancer Network (NCCN) guideline for pancreatic cancer (version 1. 2011) [Tissue samples were obtained from patients undergoing surgery at the Pancreatic Disease Institute, Union Hospital , from July 2011 to July 2012. During these procedures, 30 PC, 10 CP and 30 NP were collected. Those patients were treated with pancreatectomy or palliative surgery including implantation of I1. 2011) . 30 NP s2 at 37°C and grown in complete medium, which was composed of 90% RPMI-1640 (HyClone), 10% fetal bovine serum (HyClone) and 100 U/mL penicillin and 100 mg/ml streptomycin (HyClone). Pancreatic cancer cell lines, PANC-1 and BXPC-3, were bought from American Type Culture Collection . They were tested and authenticated for genotypes by DNA fingerprinting. These cell lines were passaged for less than 6 months after resuscitation, and no re-authorization was done. In order to build the hypoxia model, cells were cultured with 1% O2, 5% CO2 and 94% N2 or treated with CoCl2 (100 μM) [Cells were incubated in 5% CO(100 μM) . Total RTotal RNA was extracted from tissue samples and cleaned up firstly. Then prepare labeling reaction by using Quick Amp Labeling Kit, One-Color (Agilent). These cDNA samples were then cleaned and labeled in accordance with the Agilent Gene Expression Analysis protocol using Low Input Quick-Amp Labeling Kit, one-color (Agilent p/n 5190-0442). These labeled cDNA samples were purified and further used as probes to hybridize to microarrays for 17 h at 65°C using an Agilent Gene Expression Hybridization Kit (Agilent p/n 5188-5242) in hybridization chamber gasket slides (Agilent p/n G2534-60003). After hybridization, the microarrays were washed and scanned with an Agilent microarray scanner (Agilent p/n G2565BA).Markov cluster algorithm (MCL) was used to identify co-expressed functional module in the CNC network. Moreover, we identified well-established miRNAs that bind lncRNAs by using an algorithm named miRcode that predicts putative microRNA binding sites in lncRNAs using criteria such as seed complementarity and evolutionary conservation.LncRNA-NUTF2P3-001-siRNA (NUTF2P3-001-siRNA), lncRNA-NUTF2P3-001 pcDNA3.1 plasmid (pcDNA-NUTF2P3-001), miR-3923 mimics (miR-3923), miR-19b-3p mimics (miR-19b-3p), miR-3923 inhibitor (inh-3923), HIF-1α-siRNA and corresponding negative control were purchased from Ribobio Co. . NUTF2P3-001-siRNA, HIF-1α-siRNA, miR-19b-3p and miR-3923 were transfected with Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM, while the final concentration for inh-3923 was 100 nM and all plasmids were transfected with 0.2 μg for 96 well plate and 1.6 μg for 12 well plate. The effects of the transient transfection were confirmed by quantitative real-time PCR analysis. We extracted protein and total RNA 48 h post-transfection. All siRNA sequences and NUTF2P3-001 clone sequence in pcDNA3.1 are shown in Lentivirus vector (GV115) containing NUTF2P3-001-siRNA sequence (LV-NUTF2P3-001-siRNA), LV-miR-3923 and corresponding negative control (LV-NC-siRNA and LV-miR-NC) were purchased from GeneChem Company . According to the results of preliminary experiment, MOI (multiplicity of infection) values of PANC-1 and BXPC-3 were 40 and 80 respectively. Cells were cultured in ENi.S. (Enhanced infection Solution) with Polybrene (5 μg/ml) and lentivirus. 12 hours later, the mix was changed with complete medium. At last, we identified the transfection efficiency by observing fluorescence under fluorescence microscope 3 days later.Western blot analysis has been done as described previously . Antibod® RT Master Mix Perfect Real Time (TAKARA) and One Step PrimeScript® miRNA cDNA Synthesis Kit  (TAKARA), followed by qRT-PCR analysis with the SYBR Premix Ex Taq II (TAKARA) as the manufacturer's protocol. All reactions were performed in triplicate. The primers were shown in Table Total RNA was extracted from tissues and cells by using RNAiso Plus (TAKARA) according to the product description. QRT-PCR was used to check the expression level of mRNAs. All mRNAs and miRNAs were reverse transcribed according to the protocol of the PrimeScript3 cells were seeded in new 96-well plate with complete medium, the viability was assessed after 2 days and the proliferation was observed for 7 days. Cells were cultured with 20 μl MTT (5 mg/ml) per well for 4 h in incubator. Then replaced the mix of MTT and medium with 150 μl dimethylsulfoxide (Sigma) each well. Finally, determined the absorbance by ELISA reader at 570 nm when the crystals were totally dissolved. All MTT assays were repeated three times in five replicates.After stable transfection, 2.5 × 104 cells were placed in the upper chamber with 200 μl medium containing 0.1% serum. Then 700 μl medium containing 30% serum was placed in the lower chambers [We maintain the cells' invasion capacity by using the Matrigel Invasion Chamber of pore size 8 μm . The chambers were coated with 1:9 diluted Matrigel (Sigma) for 4 hours, 5 × 10chambers , 44. Fix6 per ml with 500ul binding buffer, followed the instruction protocol, cells were stained with Annexin V-FITC (5 μl) and PI  for 15 mins at 4 degrees. For cell cycle analysis, prior to treat with PI (50 μg/ml) and RNase-A (25 μg/ml) for 15 mins, cells were fixed in 70% cold ethanol at −20°C overnight. Cells were analyzed by a FACS Calibur flow cytometer (BD Biosciences) and data were analyzed with Flowjo software. All of these were repeated in duplicates.For cell apoptosis analysis, diluted the cells to 10For KRAS mRNA 3′UTR luciferase reporter assays, luciferase reporter plasmid (100 ng) containing the potential binding sequence of 3′UTR of KRAS mRNA  or mutated sequence  were co-transfected into PANC-1 cells in 96-well plate with miR-3923 (50 nM), inh-3923 (100 nM), NUTF2P3-001-siRNA (50 nM), pcDNA-NUTF2P3-001 (0.2 μg), and corresponding NC respectively by using Lipofectamine 2000 (Invitrogen). For lncRNA-NUTF2P3-001 promoter luciferase reporter assays, luciferase reporter plasmid (100 ng) containing the potential HRE1 sequence  or mutant sequence  were co-transfected into PANC-1 cells in 96-well plate with HIF-1α-siRNA (50 nM) and corresponding NC. Luciferase activity assays were performed 48 hours after transfection . Firefly luciferase activity was normalized to the corresponding renilla luciferase activity by using the Dual-Luciferase Reporter Assay System. All experiments were performed three times.tm Chromatin Immunoprecipitation Kit and anti-HIF-1α  according to the manufacturer's instructions. Corresponding IgG was used as controls. The bound DNA fragments were subjected to PCR reactions using the primers in Table ChIP assays were performed by using EpiQuik7 in 200 μl PBS) were injected subcutaneously into the right flank of 3-week-old male BALB/c nude mice (n = 5 per group). The tumor size was measured every 3 days and calculated by the formula: tumor volume = length × width2/2 [Stable transfected PANC-1 cells . Unpaired


Drosophila optomotor-blind (omb) and human TBX2 in the epithelium of the Drosophila wing imaginal disc and observed two types of cell motility. Omb/TBX2 overexpressing cells could move within the plane of the epithelium. Invasive cells migrated long-distance as single cells retaining or regaining normal cell shape and apico-basal polarity in spite of attenuated apical DE-cadherin concentration. Inappropriate levels of DE-cadherin were sufficient to drive cell migration in the wing disc epithelium. Omb/TBX2 overexpression and reduced DE-cadherin-dependent adhesion caused the formation of actin-rich lateral cell protrusions. Omb/TBX2 overexpressing cells could also delaminate basally, penetratingthe basal lamina, however, without degradation of extracellular matrix. Expression of Timp, an inhibitor of matrix metalloproteases, blocked neither intraepithelial motility nor basal extrusion. Our results reveal an MMP-independent mechanism of cell invasion and suggest a conserved role of Tbx2-related proteins in cell invasion and metastasis-related processes.The transcription factors TBX2 and TBX3 are overexpressed in various human cancers. Here, we investigated the effect of overexpressing the orthologous Tbx genes  The closely related T-box transcription factors TBX2 and TBX3 are frequently overexpressed in melanoma -3 and vaTBX2 and TBX3 are members of the Tbx2 subfamily of T-box transcription factors . They arDrosophila, the only Tbx2 subfamily gene is optomotor-blind (omb). Like its vertebrate orthologs, Omb controls many developmental processes , ) was co-ion Fig.  nor peneion Fig. . Such diion Fig. . Expression Fig. . In ordeed (puc)  under coed (puc)  and was ed (puc) , 63. Howp35 . It. ItDrosoIn recent years, the traditional view of metastasis as an endpoint of tumor progression, reached by the accumulation of clonally selected mutations, has been challenged. There are indications that cancer cells may be able to leave the primary tumor early in progression , 126-129Expression and activity of TBX2/3 are controlled by several signaling pathways , 134-139http://flybase.bio.indiana.edu unless indicated otherwise. Larvae were reared at 25°C. Larvae containing Gal80ts-Gal4 combinations were raised at 18°C and then shifted to 29°C for UAS transgene expression. Transgenes used were UAS-GFP, UAS-bsk[DN] [http://flytrap.med.yale.edu). dpp-Gal4 was obtained from K. Basler.All the stocks are described at -bsk[DN] , UAS-CD8-bsk[DN] , 30A-Gal-bsk[DN] , AYGal4 -bsk[DN] , C765-Ga-bsk[DN] , tubP-Ga-bsk[DN] , UAS-omb-bsk[DN] , UAS-DE--bsk[DN] , UAS-DIA-bsk[DN] , puc-lac-bsk[DN] . Viking-ccdB, and attR2, was obtained from pT-REx-DEST30 (Invitrogen) by BclI linker amplification. This cassette was cloned into the BglII site of pCa4B2G-UAS resulting in pCa4B2G-UAS-GW. The human TBX2 open reading frame was amplified from a cDNA clone  by linker-PCR which appended CACC at the 5′ end for directional cloning and and a triple FLAG-tag at the 3′ end . Because of the high GC content, amplification was perfomed with GC-rich PCR System (Roche). The amplified fragment was polished with PfuUltraTM High-Fidelity DNA polymerase using the PCR Polishing kit (Stratagene) and directionally cloned into pENTRTM/D-TOPO® using the pENTRTM Directional TOPO® Cloning Kit (Invitrogen). From this entry clone the tagged TBX2 sequence was transferred to the destination vector pCa4B2G-UAS-GW by LR recombination using Gateway® LR ClonaseTM II emzyme mix (Invitrogen) to obtain TBX2-3xFLAG/pCa4B2G-UAS-GW. Transgenic flies were obtained by phiC31 recombination at attP landing site 58A (Rainbow Transgenic Flies).We first constructed a Gateway® (Invitrogen) compatible UAS vector based on the vector pCa4B2G . The UASMarked clones of mutant cells were generated by Flp-mediated mitotic recombination  subjectiDissected wing imaginal discs were fixed and immunostained using standard procedures for confocal microscopy. Imaginal discs dissected from third instar larvae were fixed and stained with rhodamine phalloidin , DAPI , ECM-P1  and apprThird instar larvae were dissected in ice-cold 1x PBS and fixed in 4% paraformaldehyde. After several washing steps with PBTween, larvae were incubated in 1ml 1.2 M triethanolamine, pH 7,0 mixed with 2,5 μl acetic anhydride for 1 hour and then prehybridized for 4 hours at 65°C. Larval tissue was hybridized at 65°C overnight with digoxigenin-labeled RNA antisense probes that were generated using a PCR-amplified template tagged with a T7-polymerase (Roche) binding site.The next day, anti-DIG-antibody labelled with alkaline phosphatase (Roche) was diluted 1 to 1000 and incubated with larval tissue for 2 hours at room temperature. The staining was performed with 3,3 μl NBT and 1,5 μl BCIP solutions in 1ml AP-buffer. To stop the staining, tissue was washed for 10 min in methanol. For further handling, larvae were stored in 70% glycerol in PBS.


Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists , which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents ). Using  in vitro cellular models of HIV-1 latency as well as in ex vivo primary cell cultures from cART-treated HIV+ aviremic patients identified bryostatin-1+JQ1 and ing-B+JQ1 to potently reactivate latent HIV-1. The potent effects of these two combinations were detected as early as 24 hours post-treatment. Importantly, bryostatin-1 was used at concentrations below the drug plasma levels achieved by doses used in children with refractory solid tumors. Our mechanistic data established a correlation between potentiated P-TEFb activation and potentiated or synergistic (depending on the HIV-1 latency cellular model used) induction of HIV-1 gene expression observed after the combined versus individual drug treatments. In conclusion, our results establish a proof-of-concept for PKC agonists combined with compounds releasing active P-TEFb as a strategy proposed for a cure or a durable remission of HIV infection.Persistence of latently infected cells during cART is a major hurdle for HIV-1 eradication. A widely proposed strategy to purge these reservoirs involves the reactivation of latent proviruses. The low levels of active P-TEFb and the cytoplasmic sequestration of NF-κB in resting infected cells largely contribute to maintenance of HIV-1 latency. Therefore, utilization of chemical compounds that target both pathways may lead to more potent effects on HIV-1 reactivation than the effect mediated by the individual drug treatments. In this study, we showed that combined treatments of PKC agonists  with compounds releasing P-TEFb  exhibited a synergistic increase in viral reactivation from latency. In-depth comparison of combined treatments in various  In conc+ patients .CD8escribed  66]. Re. Re+-dep+-depleted PBMCs or resting CD4+ T cell ex vivo cultures using the QIA amp DNA Mini or the QIA amp Micro kit (Qiagen), respectively. The total cell-associated HIV-1 DNA was then quantified by ultra-sensitive real-time PCR  accordi+-depleted PBMCs or patient resting CD4+ T cell ex vivo cultures were collected for RNA extraction using QIA amp Viral RNA Mini kit (Qiagen). HIV-1 RNA levels were quantified using the Generic HIV Charge Virale kit (Biocentric) according to the manufacturer’s instructions (detection limits of 150 HIV-1 RNA copies/ml or 15 HIV-1 RNA copies/ml depending on tested supernatant volumes).Six days after drug treatment, culture supernatants from either patient CD8The outgrowth viruses, previously quantified by the determination of extracellular HIV-1 RNA concentration, were concentrated by ultracentrifugation of 3ml of culture supernatants collected 6 days post-treatment and resuspended in 50μl of culture medium. Infections of Jurkat cells were performed by resuspending 50 thousand cells in 50μl of the outgrowth viral stocks and incubating at 37°C for 2 hours.The multiplicity of infection was in the range of 0.02–0.05. After infection, the cells were pelleted at 300xg, washed three times with 200μl of culture medium, resuspended in 200μl of culture medium, and grown under standard conditions in 96-well plates. Two days post-infection, cells were amplified by adding 400μl of culture medium and seeded in 24-well plates. Six days post-infection, 350μl of culture supernatants were collected for RNA extraction using QIAamp Viral Mini Kit (Quiagen) according to manufacturer’s instructions. HIV-1 RNA levels were quantified using the Generic HIV Charge Virale kit (Biocentric) (detection limit of 75 HIV-1 RNA copies/ml).+-depleted PBMCs or resting CD4+ T cells isolated from blood of uninfected donors were used to establish ex vivo culture cells. Cells were collected 6 days after stimulation with different LRAs and were stained with relevant antibodies. All antibodies were purchased from BD Biosciences and included three antibody cocktails. The first cocktail included anti-CD4 (557852), anti-CD8 (345774) and anti-CD38 (345806). The second cocktail included anti-CD4 (345770), anti-CD8 (348813), anti-HLA-DR (347401), anti-CD69 (347823) and anti-CD25 (340907). The third cocktail included anti-CD4 (345770), anti-CD8 (345772), anti-CD38 (555462) and anti-HLA-DR (347401). Marker staining was assessed by flow cytometry analysis on a FACS CantoII (Becton-Dickinson) and analyzed using the FACSDiva Software (Becton-Dickinson).For cell activation analysis, CD8ex vivo reactivation studies using patient cell cultures of CD8+-depleted PBMCs or of resting CD4+ T cells are shown as means. Data sets were analyzed using an unpaired, nonparametric Mann-Whitney U test. P<0.05 was considered statistically relevant. Analyses were performed using Prism version 6.0 (GraphPad software).T-cell activation data, S1 FigA, C, E, and G) and U1  cell lines were mock-treated or treated with increasing doses of compounds as indicated. At 24 hours post-treatment, CA-p24 production in cell supernatants  or cell metabolic activity  were measured. Results obtained with the mock-treated cells were arbitrary set at a value of 1 or 100%, respectively. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented.The J-Lat 9.2 (panels (PPT)Click here for additional data file.S2 FigOM10.1 cells were mock-treated or treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5Mm), bryostatin-1 (10nM) and prostratin (2.5 μM) alone or in combination as indicated. At 24 hours post-treatment, CA-p24 production in cell supernatants were measured. Results obtained with the mock-treated cells were arbitrary set at a value of 1 or 100%, respectively. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from two is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.(PPT)Click here for additional data file.S3 FigA), J-Lat cell line A2  or A72 (panel C) containing a stably integrated LTR-GFP construct were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5 μM) alone or in combination as indicated. At 24 hours post-treatment, cells were analyzed by flow cytometry to quantify the proportion of cells expressing GFP. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from two is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.The THP89GFP cells (panel (PPT)Click here for additional data file.S4 FigA), CHME-5/HIV microglial cells (panel B) or THP89GFP monocytic cells (panel C) harbor latent HIV1 provirus containing gfp gene. The cells were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5 μM) alone or in combination as indicated. At 24 hours post-treatment, cells were analyzed by flow cytometry and the mean fluorescence intensity (MFI) was analyzed to quantify the amount of GFP produced. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.The J-Lat 9.2 cell line (panel (PPT)Click here for additional data file.S5 Figex vivo cultures of CD8+-depleted PBMCs isolated from blood of 5uninfected donors were incubated with indicated compounds for 6 days. The result obtained with mock-treated cells was set at a value of 100%.WST-1 assay on (PPT)Click here for additional data file.S6 FigA. WST-1 assay on ex vivo cultures of CD8+-depleted PBMCs isolated from blood of 4 uninfected donors were incubated with indicated compounds for 6 days. The result obtained with mock-treated cells was set at a value of 100%. Panel B. Cell viability. Trypan blue exclusion assay was performed on the same patient cell cultures as described in (A).The result obtained with mock-treated cells was set at a value of 100%.Panel (PPT)Click here for additional data file.S7 Fig+-depleted PBMCs from 4 uninfected donors were mock-treated, treated with anti-CD3+anti-CD28 antibodies (C+), JQ1 (0.25μM), bryostatin-1 (5nM), prostratin (0.5μM) or ingenol B (10nM) alone or in combination for 6 days. Cells were incubated with anti-CD38, anti-HLA-DR, anti-CD4 and anti-CD8 antibodies prior to flow cytometry analysis. The results are presented as percentage of marker expression in the population of CD4+ cells. Dashed line indicates the percentage of expression obtained in mock-treated cells. The means are represented.CD8(PPT)Click here for additional data file.S1 Table+T cell count, CD4+ nadir, antiviral regimens, duration of therapy, duration with undetectable plasma HIV-1 RNA level, and HIV-1 subtypes) of patients from the St- Pierre Hospital are presented. “X” indicates not reported.Characteristics Click here for additional data file.S2 TableEx vivo cultures of CD8+-depleted PBMCs from blood of 3 cART-treated HIV+ patient were treated with bryostatin-1+JQ1 for 6 days. Concentrations of viral RNA in culture supernatants were measured and were expressed as HIV-1 RNA copies/ml. Total HIV-1 DNA was expressed as HIV-1 DNA copies/106 CD8+-depleted PBMCs. Viruses collected from ex vivo cell cultures were used to perform de novo infection of Jurkat cells. Six days post-infection, Jurkat culture supernatants were collected and concentrations of viral RNA in culture supernatants were quantified and expressed as HIV-1 RNA copies/ml).(PPT)Click here for additional data file.


HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis.Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development.  trans-Golgi network/early endosome to the plasma membrane was disrupted in hap13-1, a mutant of HAPLESS13. We further demonstrate that defects of SUB targeting in the outer integuments compromised FG development, suggesting an intriguing intercellular communication. Our results highlight the importance of vesicular trafficking in the establishment of ovular polarity and in the communications between spatially separated cells.Ovules, being the progenitors of seeds, hold an important role in crop production. Ovule is a complex organ connecting two generations, i.e. diploid sporophytic cells and haploid female gametophytes (FG). Fertilization of female gametes leads to the development of embryo and endosperm while the sporophytic cells of ovules become seed coats. Because of its importance for plant reproduction, the development of ovules is tightly controlled. The integument cells are divided in a polar way so that integument cell layers fully enclose a mature FG, leaving an opening only for pollen tube entrance. The integuments also instruct FG development, synchronizing the two generations. Receptor-like kinases, especially STRUBBELIG (SUB), are crucial both for the establishment of ovular polarity and the intercellular communication between sporophytic and gametophytic cells. However, how SUB is post-translationally regulated, as would be expected for cell-surface proteins, is unclear. We report here that the dynamic trafficking of SUB via a subpopulation of endosomes is crucial for its functionality during ovule development. The membrane association of SUB was impaired when the recycling route from the  Tissue or planar polarity that distinguishes multicellular organisms from unicellular ones requires coordinated cell behavior within a tissue or the plane of a tissue layer through intercellular communications and intracellular asymmetry ,2. OvulePIN1-PIN8), whose dynamic distribution, often asymmetric, is essential for auxin gradient [STRUBBELIG (SUB), ARABIDOPSIS CRINKLY4 (ACR4), and ERECTA-family genes (ER and ERLs), resulted in defective ovular development [Intercellular communication in plant development is mainly controlled by the phytohormone auxin as well as receptor-like kinases (RLKs) ,8,9. Auxgradient ,11. Amongradient ,13. Tisselopment –16.The membrane distribution of PINs and RLKs is dynamically regulated by vesicle trafficking-mediated protein sorting ,17–19. Itrans-Golgi network/early endosome (TGN/EE) [AP1μ, HAPLESS13 (HAP13)/AP1MU2 [HAP13 severely reduced male gametophytic transmission [HAP13, hap13-1, demonstrating the key role of HAP13 in TGN/EE-centered protein sorting.An exocytic or anterograde trafficking route and an endocytic or retrograde trafficking route operate in plant cells to mediate protein sorting within the endomembrane system –22. The (TGN/EE) , a likel(TGN/EE) ,22. Prot(TGN/EE) ,22,24. A(TGN/EE) –27. AP1 (TGN/EE) ,22,24. W)/AP1MU2 –27. Mutasmission ,27,28 whsmission ,27, BRI1smission  were mishap13-1 is defective in outer integument growth, leading to exposed or protruding nucellus. The defect in outer integuments severely affected female gametogenesis such that a large portion of hap13-1 ovules did not contain a well-patterned embryo sac. Consequently, pollen tube guidance and reception were impaired, resulting in reduced female fertility. We further show that the key RLK for ovule development, SUB, was mis-targeted in hap13-1, correlating with the phenotypic similarity in ovules between hap13-1 and SUB mutants. However, unlike PIN2 whose membrane distribution relies on HAP13 [hap13-1, suggesting distinct sorting mechanisms for different PINs. Downregulating HAP13 expression by RNA interference (RNAi) specifically in outer integuments phenocopied the ovular defects in hap13-1. Results presented here demonstrate the importance of TGN/EE-centered protein sorting in outer integument growth and provide clues for a deep understanding of molecular mechanisms underlying the establishment and maintenance of ovule polarity.We report here that HAP13-mediated protein sorting at the TGN/EE is critical for ovule development, specifically the asymmetric growth of outer integuments. By scanning electron microscopy (SEM), plastic section, and confocal laser scanning microscopy (CLSM), we show that on HAP13 ,27, the HAP13 was expressed in various tissues and developmental stages [HAP13 was expressed during ovule development, we generated a nuclear-localized YFP reporter line for HAP13 (ProHAP13:NLS-YFP). Confocal laser scanning microscopy (CLSM) revealed that HAP13 was expressed in all cell layers during ovule development , pollen tubes already entered the transmitting track in wild-type pistils but only exited the style in hap13-1 pistils  showed a substantial reduction of fluorescence signals . Developmental defects of hap13-1 started at stage 2-III  verified a significant reduction of HAP13 transcripts by ProUBQ10:HAP13-RNAi . ProINO was reported previously for its specific expression in ovules based on RNA in situ hybridization [ProINO was specific for outer integuments where severe growth defects were observed in hap13-1, we performed histochemical GUS staining on ProINO:GUS transgenic ovules at different stages. GUS signals were first observed at the stage 2-I in cells to be differentiated into outer integuments  were used for further analysis by CLSM and SEM. SEM of developing ovules revealed that HAP13-RNAi resulted in severe reduction of outer integument growth -primed cDNAs were synthesized using Superscript III reverse transcriptase with on-column DNase II digestion (Invitrogen). Real time quantitative PCRs were performed as described [Total RNAs for detecting escribed . PrimersProINO (2012 bp before the start codon of INO); ZP96/ZP97 for ProDD45 (1003 bp before the start codon of DD45). The destination vector used to generate ProDD45:GUS and ProINO:GUS were described previously [ProUBQ10:HAP13-RNAi construct. Later, ProUBQ10 was replaced with ProINO to generate ProINO:HAP13-RNAi. A ProINO:NLS-YFP expression cassette was amplified with primers ZP3872/ZP3873 containing the restriction enzyme site SacI and EcoRI, respectively. The PCR fragment was inserted into the expression vector to generate the ProINO:HAP13-RNAi ProINO:NLS-YFP construct. Primers are listed in All constructs were generated using the Gateway technology (Invitrogen) except the RNAi construct. Entry vectors were generated using pENTR/D/TOPO (Invitrogen). Coding sequences or promoter sequences were cloned with the following primer pairs: ZP1781/ZP1782 for eviously . Expresseviously  to obtaiHistochemical GUS staining, ovule whole-mount clearing, semi-thin sections, and SEMs were performed as described . Aniline2, 1 mM MgSO4, 50 mM HEPES (pH 7.4), 5% sucrose, pH8.0 with NaOH) for 10 min with vacuum desiccation at room temperature (RT). The fixative solution was then removed and pistils were washed three times for 10 min each with the PME buffer . The fixed pistils were treated further for permeation sequentially with a PME buffer, cellulose solution, and 3% IGEPAL CA-630. After permeation, pistils were sequentially incubated with the primary antibody  and second antibody , followed by tissue mounting and CLSM examination. Uptake of FM4-64, BFA treatment and CLSM were performed as described [Whole-mount immuno-fluorescence staining of ovules using an anti-LURE antibody  was perfescribed . OpticalHEPES pH .4, 5% suescribed .HAP13, At2g21750 for DD45, At1g23420 for INO, At5g43510 for LURE1.2.Sequence data from this article can be found in the GenBank databases under the following accession numbers: At1g60780 for S1 Fighap13-1 (B), or homozygous hap13-1 (C) pistils transformed with ProDD45:GUS. Percentage of ovules showing GUS signals is shown on top of each image. Results are means ± standard error (s.e.m). In total, 18 pistils were analyzed for each genotype. Bars = 200 μm.(A-C) Histochemical analysis of wild-type (A), heterozygous (TIF)Click here for additional data file.S2 Fighap13-1 (B) with the same illumination. The output images have not been brightened. Bars = 10 μm. (C) Relative transcript abundance of SUB by quantitative real-time PCRs. Results shown are means ± s.e.m. Three biological replicates were analyzed. No significant difference was observed .(A-B) CLSM of a representative stage 2-III ovule from SUBg-YFP in wild type (A) or in (TIF)Click here for additional data file.S3 Fighap13-1 seedlings. Con, controls; WO, washout. Arrows point at BFA compartments. Bars = 10 μm.CLSM of SUBg-YFP in roots of 4 DAG wild-type or (TIF)Click here for additional data file.S4 Fighap13-1 seedlings  upon 1 min FM4-64 uptake , upon BFA treatment for 50 min , or after BFA washout . Bars = 25 μm.(A-F) CLSM of PIN1:GFP in roots of 4 DAG wild-type  or (TIF)Click here for additional data file.S5 FigProUBQ10:HAP13-RNAi transgenics  upon 1 min FM4-64 uptake followed by BFA treatment for 50 min  or after BFA washout . BFA washout in wild type (WT BFA wo) leads to tonoplast targeting of FM4-64 as seen by reduced signals at BFA compartments while in ProUBQ10:HAP13-RNAi transgenics (HAP13-RNAi BFA wo) FM4-64 is still largely trapped in BFA compartments. (E) Relative transcript abundance of HAP13 in wild type or six ProUBQ10:HAP13-RNAi transgenic lines by quantitative real-time PCRs. RNAs were extracted from 4 DAG seedlings. Results shown are means ± s.e.m. (N = 3). Means with different letters are significantly different . Bars = 10 μm.(A-D) CLSM of 4 DAG roots of wild type  or of (TIF)Click here for additional data file.S6 FigProINO:GUS pistils at various developmental stages. Arrowheads point at outer integuments. Bars = 20 μm for (A-C); 10 μm for (D-F).(A-F) Transverse sections of (TIF)Click here for additional data file.S7 FigProINO:HAP13-RNAi;ProINO:NLS-YFP transgenic plants. Shaded areas are either outer integuments (oi) or inner integuments (ii). The bright-field and YFP channel overlays are side-by-side with their corresponding YFP channel images. Hardly any signal was detected in the inner integuments. Bars = 20 μm.Representative severely (A) or mildly (B) affected ovules from the (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Using KrasLA1 mice, we show that TBI promotes the expansion of a rare population of thymocytes that express oncogenic KrasG12D. However, blocking p53 during TBI significantly suppresses the expansion of KrasG12D-expressing thymocytes. Mechanistically, bone marrow transplant experiments demonstrate that TBI activates p53 to decrease the ability of bone marrow cells to suppress lymphoma development through a non-cell-autonomous mechanism. Together, our results demonstrate that the p53 response to acute DNA damage promotes the development of radiation-induced lymphoma.Genotoxic cancer therapies, such as chemoradiation, cause haematological toxicity primarily by activating the tumour suppressor p53. While inhibiting p53-mediated cell death during cancer therapy ameliorates haematologic toxicity, whether it also impacts carcinogenesis remains unclear. Here we utilize a mouse model of inducible  p53 can be activated by oncogenic stress to suppress tumourigenesis, but its role in radiation carcinogenesis has not been studied in p53 wild-type mice. Here, Lee et al. show that knocking down p53 during total-body irradiation not only reduces acute toxicity, but prevents the formation of radiation-induced lymphoma. However, these mice showed no difference in the latency of lymphoma formation regardless of when p53 was deleted. These results demonstrate that the acute p53 response to radiation is dispensable for suppressing the development of lymphomas in mice that have permanently lost p53.However, mice with germline deletion of p53 mutations. Instead, they acquire somatic mutations during cell divisionp53, we use transgenic mice that harbour an inducible in vivo shRNA against p53 TRE-p53.1224 mice were crossed to either cytomegalovirus (CMV)-reverse tetracycline-controlled transactivator (rtTA)Actin)-rtTACMV-rtTA; TRE-p53.1224 and Actin-rtTA; TRE-p53.1224 mice with a Dox-containing diet for 10 days induced p53.1224 shRNA that caused a corresponding decrease in p53 mRNA expression in the bone marrow. Withdrawal of Dox decreased expression of p53.1224 shRNA and p53 mRNA was restored within 7 days  compared with littermates that contain only an rtTA or a TRE-p53.1224 allele  cells, which are enriched for haematopoietic stem/progenitor cells (HSPCs)To temporarily and reversibly control p53 expression n 7 days . TreatmeCMV-rtTA; TRE-p53.1224 and Actin-rtTA; TRE-p53.1224 mice as well as their littermate controls were fed a Dox-containing diet for 14 days. On the final 4 days, the mice received daily fractions of 1.8 Gy TBI , flow cytometry analysis revealed expansion of CD4+CD8+ double positive cells (n=5), CD4 single positive (CD4SP) cells (n=2) or CD8 single positive (CD8SP) cells  locus showed that most radiation-induced lymphomas contained only one or two dominant rearrangements , suggestlls n=2; . Remarkalls n=2; . Howeverlls n=2; . Moreovells n=2; . CompareKrasLA1 miceKrasLA1 allele has undergone intrachromosomal recombination to express oncogenic KrasG12D. In the absence of radiation, knockdown of p53 by Dox treatment for 14 days did not influence the penetrance or latency of thymic lymphoma development  compared with littermate controls  and KrasLA1; control (n=14) mice. We found that temporary knockdown of p53 did not significantly influence the survival of mice that succumbed to lung cancers  cells+ cells in peripheral blood (PB) 4–16 weeks after bone marrow transplant  cells36in vitro colony formation assays  can be inhibited after irradiation by transplantation with bone marrow cells from various donors. Donor mice (CD45.1) included unirradiated shp53 or control mice (no TBI), shp53 mice 4 days after 2.5 Gy TBI (shp53 TBI) or control mice 4 days after 2.5 Gy TBI  was performed to compare the means of two groups. Two-way analysis of variance was performed to examine the interaction between genotypes and radiation treatment. Fisher's exact test was performed to analyse data from the TCRβ locus rearrangement experiment. For carcinogenesis and haematopoietic acute radiation syndrome studies, Kaplan–Meier analysis was performed followed by the log-rank test. Significance was assumed at P<0.05. GraphPad Prism 5  was used for the calculation of the statistics.For all experiments, data are presented as mean or mean±s.e.m. and each data point represents one mouse. Where variance was heterogeneous, data were log-transformed before applying statistical tests. Student's LA1 and p53−/− mice171826PtprcaPepcb/BoyJ (CD45.1) mice were purchased from The Jackson Laboratory. Four to 8-weeks-old male and female mice were used in the study. Mice on a mixed genetic background were used for all experiments except for BMT experiments. To perform BMT experiments, CMV-rtTA; TRE-p53.1224 mice were backcrossed to C57BL/6 J mice (CD45.2) for five generations, to B6.SJL-PtprcaPepcb/BoyJ mice (CD45.1) for at least two generations, and then maintained on a CD45.1 background because we observed that the TRE-p53.1224 allele co-segregates with the CD45.1 allele. For experiments conducted using mice on a mixed genetic background, age-matched littermate controls were utilized to minimize the effect of genetic background. Therefore, potential genetic modifiers of the response to radiation would be randomly distributed among the experimental and control groups.All animal procedures for this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University. All of the mouse strains used in this study have been described previously including TRE-1224 (TRE-p53.1224 A), CMV-rtTA, Actin-rtTA, Kras−1 Dox, Cat. No. TD.04580, Harlan Teklad) for 10 days before irradiation, and were maintained on the Dox-containing diet until they received the last dose of radiation. Immediately after the last dose of irradiation, all mice were switched to a regular (Dox-free) diet. For unirradiated control studies, all mice were fed the Dox-containing diet for the same period of time as the irradiated groups and then switched to a regular (Dox-free) diet.For radiation studies, all mice were fed a Dox-containing diet . The dose rate was measured with an ion chamber by members of the Radiation Safety Division at Duke University.TBI was performed 50 cm from the radiation source with a dose rate of 200 cGy min250 μg of dexamethasone sodium phosphate (Sigma-Aldrich) in PBS was intraperitoneally injected into each mouse 30 min after irradiation. Age-matched littermates in the same cage were randomly selected to receive either vehicle or dexamethasone.Tissue specimens were fixed in 10% neutralized formalin overnight, preserved in 70% ethanol, and then embedded in paraffin. Tumour diagnosis on haematoxylin and eosin-stained sections was performed by two veterinary pathologists (Y.K. and L.B.) blinded to the genotype or treatment. Immunohistochemistry staining was performed on tissue sections using anti-mouse CD3 Proteins were extracted from cells or tissues using RIPA lysis buffer (Sigma-Aldrich). The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). A total of 30 mg of total protein were loaded for electrophoresis into 4–20% sodium dodecyl sulfate polyacrylamide gels (Bio-Rad). Separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked with 5% BSA in tris-buffered saline (TBS) with 0.1% Tween 20. Protein levels were detected using antibodies against cleaved Notch1 p53, Mm00432448_m1 for Cdkn1a, Mm00449846_m1 for Phlda3, Mm01281680_m1 for Lmo2, Mm01342805_m1 for Hes1 and Mm99999915_g1 for Gapdh). Gapdh was used as an internal control to correct for the concentration of RNA in different samples. Expression of miR-30-based p53.1224 shRNA was detected using a Custom TaqMan MicroRNA Assay (Applied Biosystems). A reverse transcription primer and a TaqMan probe were designed to specifically detect p53.1224 siRNAIn vitro reverse transcription was performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). SnoRNA 202 was used as an internal control to correct for the concentration of RNA in different samples. Each experiment was performed with three replicates from each sample, and the results were averaged.Total RNA was extracted from samples using TRIzol reagent (Life technologies) and reverse transcription was performed using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative reverse transcription-PCR (qRT–PCR) was performed to detect mRNA expression using Taqman Universal Mix II (Applied Biosystems) with Taqman probes . The 3.5 kb PCR amplicon was purified using QiAquick PCR Purification Kit (Qiagen) and then used as a template for the subsequent droplet digital PCR experiment. For droplet digital PCR, we used the Primer Express software (Applied Biosystems) to design VIC and fluorescein amidite (FAM) conjugated Taqman MGB probes to specifically detect the WT Kras allele and the KrasG12D mutant allele, respectively. The sequences of primers and probes for these two assays were:Genomic DNA was extracted from thymocytes or lymphomas using DNeasy Blood & Tissue Kit (Qiagen) and quantified using NanoDrop (Thermo Scientific). To assess the frequency of the recombined Forward primer: 5′-TGTAAGGCCTGCTGAAAATGACT-3′Reverse primer: 5′-TGTATCGTCAAGGCGCTCTTG-3′WT probe: 5′-TTGGAGCTGGTGGC-3′Kras G12D probe: 5′-TTGGAGCTGATGGC-3′KrasDigital PCR was performed using the RainDrop Digital PCR System (RainDance Technologies) following procedures described previously+ and Mg2+, 5% fetal bovine serum, 2 mM EDTA). Red blood cells (RBC) were lysed using ammonium-chloride-potassium (ACK) lysing buffer (Lonza). Total number of thymocytes was counted with a Coulter counter (Beckman Coulter). 1 × 106 thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with phycoerythrin (PE)-Cy5 conjugated anti-mouse CD4 (clone: GK1.5), PE-conjugated anti-mouse CD8 (clone: 53-6.7), allophycocyanin (APC) conjugated anti-mouse CD44 (clone: IM7), fluorescein isothiocyanate (FITC) conjugated CD25 (clone: PC61.5) and APC-eFluor780 conjugated anti-mouse cKit (clone: 2B8) antibodies (eBioscience). All antibodies were diluted 1:400. Dead cells were excluded by staining with Calcein Blue AM (Life technologies). Data were collected from 250,000 single cells by FACSCanto (BD Pharmingen) and analysed by FlowJo  without knowledge of the genotype or treatment by a single observer (C.L.L.).Total thymocytes were isolated from the thymus in haematopoietic stem cell (HSC) buffer . Total number of WBM cells was counted with a Coulter counter (Beckman Coulter). Three million WBM cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated anti-mouse CD3e (clone: 145-2C11), PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5), PE-Cy5 conjugated anti-mouse CD8 (clone: 53-6.7) PE-Cy5 conjugated anti-mouse B220 (clone: RA3-6B2), PE-Cy5 conjugated anti-mouse CD11b (clone: M1/70), PE-Cy5 conjugated anti-mouse Gr-1 (clone: RB6-8C5), PE-Cy5 conjugated anti-mouse Ter119 (Ly-76), PE-conjugated anti-mouse Sca1 (clone:D7), APC conjugated anti-mouse cKit (clone: 2B8), FITC conjugated anti-mouse CD48  and Brilliant Violet 421 conjugated anti-mouse CD150  antibodies. All antibodies were diluted 1:400. Dead cells were excluded by staining with 7AAD (BD Pharmingen). Data were collected from 1 million single cells by FACSCanto (BD Pharmingen) and analysed by FlowJo  without knowledge of the genotype or treatment by a single observer (C.L.L.).6 RBC-lysed WBM cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with APC conjugated anti-mouse CD45 , PE-conjugated anti-mouse CD201 , APC-eFluor780 conjugated anti-mouse CD48  and Brilliant Violet 421 conjugated anti-mouse CD150  antibodies. All antibodies were diluted 1:400. Stained cells were fixed in HSC buffer containing 2% paraformaldehyde overnight at 4 °C. Fixed WBM cells were permeabilized using HSC buffer containing 0.025% Triton X-100 followed by Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Pharmingen). Permeablized WBM cells were stained with a FITC conjugated anti-human Ki67 antibody (BD Pharmingen) and then resuspend in HSC buffer containing 7AAD (BD Pharmingen). Data were collected from 1 million single cells by FACSCanto (BD Pharmingen) and analysed by FlowJo  without knowledge of the genotype or treatment by a single observer (C.L.L.).3 × 10WBM cells and thymocytes were isolated following the same procedure described above. Cleaved caspase-3 staining was performed using PE Active Caspase-3 Apoptosis Kit (BD Pharmingen) according to the manufacturer's instructions. Data were collected from 30,000 single cells by FACSCanto (BD Pharmingen) and analysed by FlowJo  without knowledge of the genotype or treatment by a single observer (C.L.L.).in vitro colony formation assays, RBC-depleted WBM cells were isolated and counted following the same procedure described above. WBM cells were plated in MethoCult GF 3434 (Stem cell technologies) and colonies were counted 7 days later. Experiments to assess colony-forming unit-spleen 12 was performed following the protocol described previouslyFor CD45.1 donor mice were placed on Dox and treated with TBI as in CMV-rtTA; TRE-p53.1224 mice and their littermate controls on a CD45.1 background. These mice were fed a Dox-containing diet for 11 days. On day 11, mice were exposed to a single fraction of 2.5 Gy TBI or no irradiation. WBM cells were harvested from these donor mice 4 days after TBI following the protocol described above and used for the subsequent transplant experiment. Recipient mice, which were 6-week-old male C57BL/6 J (CD45.2) mice, were exposed to 4 weekly fractions of 1.8 Gy TBI to induce thymic lymphoma7 WBM cells from donor mice with various genotypes and radiation treatment via intravenous injection. At 4, 10, 16 weeks after BMT, PB was collected following the protocol described above. PB-MNCs were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with FITC conjugated anti-mouse CD45.2 , APC-eFluor780 conjugated anti-mouse CD45.1 , PE-conjugated anti-mouse B220 , PE-Cy5 conjugated anti-mouse CD11b , PE-Cy5 conjugated anti-mouse Gr-1  and Brilliant Violet 421 conjugated anti-mouse CD3e antibodies . All antibodies were diluted 1:400. Data were collected from at least 20,000 single cells by FACSCanto (BD Pharmingen) and analysed by FlowJo  without knowledge of the genotype or treatment by a single observer (C.L.L.).The purpose of this experiment was to examine if radiation-induced lymphoma in recipient mice can be suppressed by transplanting bone marrow cells from different donors33How to cite this article: Lee, C.-L. et al. Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma. Nat. Commun. 6:8477 doi: 10.1038/ncomms9477 (2015).Supplementary Figures 1-8


Their structures were elucidated by spectroscopic methods including extensive 2D NMR techniques and X-ray crystallography. All the compounds showed no significant activity against five human cancer cell lines. Four new bisabolane sesquiterpenoids daedatrins A-D (Supplementary material is available for this article at 10.1007/s13659-013-0065-0 and is accessible for authorized users. Supplementary material, approximately 5.14 MB.


After identifying a down-regulation of the miR-30 family  when compared to C57Bl/6 (WT) mice, we found that overexpression of miR-30 family miRNAs promotes differentiation, while inhibition restricts differentiation of myoblasts in vitro. Additionally, miR-30 family miRNAs are coordinately down-regulated during in vivo models of muscle injury (barium chloride injection) and muscle disuse atrophy (hindlimb suspension). Using bioinformatics tools and in vitro studies, we identified and validated Smarcd2, Snai2 and Tnrc6a as miR-30 family targets. Interestingly, we show that by targeting Tnrc6a, miR-30 family miRNAs negatively regulate the miRNA pathway and modulate both the activity of muscle-specific miR-206 and the levels of protein synthesis. These findings indicate that the miR-30 family may be an interesting biomarker of perturbed muscle homeostasis and muscle disease.microRNAs (miRNAs) are short non-coding RNAs that can mediate changes in gene expression and are required for the formation of skeletal muscle (myogenesis). With the goal of identifying novel miRNA biomarkers of muscle disease, we profiled miRNA expression using miRNA-seq in the gastrocnemius muscles of dystrophic  Skeletal muscle can be affected by a variety of genetic and acquired pathologies, in most cases leading to a loss of muscle mass. In particular, Duchenne Muscular Dystrophy (DMD) is a lethal muscle degenerative disease that affects 1 in 3500 males ), the dimicroRNAs (miRNAs) are short, endogenous non-coding RNAs that post-transcriptionally repress gene expression by promoting translational repression and/or mRNA decay, resulting in decreased protein expression . miRNAs mdx4cv gastrocnemius muscles, we found the miR-30 family miRNAs to be coordinately down-regulated when compared to WT. Given the high abundance in skeletal muscle and differential expression, we decided to further investigate the expression and function of miR-30 family miRNAs in mammalian skeletal muscle. Our results indicate that expression of the miR-30 family miRNAs is perturbed during alterations in muscle homeostasis in vivo, and that the miR-30 family miRNAs promote myoblast terminal differentiation and restrict proliferation in vitro. In addition, we identify the chromatin remodeling component Smarcd2, the transcriptional repressor Snai2 and the miRNA pathway component Tnrc6a as direct miR-30 targets. Interestingly, we found that inhibition of Tnrc6a expression by miR-30 family miRNAs reduces the activity of muscle-enriched miR-206, indicating that the miR-30 family constitutes a negative feedback mechanism on the miRNA pathway. These findings expand our understanding of miRNA-mediated gene repression.By deep sequencing small RNAs from wild-type C57Bl/6 (WT) and dystrophic mdx4cv animals  on RNA isolated from the gastrocnemius of 3-month old male WT and  animals , and tha animals . The miR animals  and like animals . In orde animals . This re animals . In huma animals , but we mdx4cv), we wanted to examine miR-30 family expression in other models of skeletal muscle pathology, including regeneration after acute injury and muscle disuse atrophy. Accordingly, we first performed barium chloride injury in the gastrocnemius muscles of WT animals to test regeneration after injury in vivo and measured miR-30 family expression on 1, 3, 7 and 14 days post-injury (DPI) in comparison to uninjured contralateral controls. Barium chloride is an established model of muscle degeneration that induces widespread necrosis of myofibers and subsequent regeneration [Given our observed decrease in miR-30 family expression in a pathological setting of constant degeneration/regeneration  composition . After b2C12 myoblast differentiation in vitro. Following withdrawal of serum from the medium, we observed increases in miR-30a-5p (∼1.5 fold), miR-30b (∼2 fold) and miR-30c (∼2 fold) expression as differentiation progressed  in the developing muscle  nuclei indicated a striking 65% increase (P = 5e-5), indicat2C12 myoblasts  . As indiyoblasts , again i2C12 with a representative synthetic miR-30 family member, miR-30a-5p, then performed 5-ethynyl-2’-deoxyuridine (EdU) proliferation analysis. In comparison to a scrambled pre-miR control at equivalent concentrations, EdU incorporation was reduced dose-dependently by 10% and 15% (P ≤ 0.05) in 10nM and 50nM transfected cells, respectively , a transcription factor that regulates mitochondrial biogenesis in muscle [Runx1 (AML-1) a subunit of the core binding factor (CBF) and transcriptional repressor of myogenesis [Smarcd2 (Baf60b), a SWI/SNF chromatin remodeling complex subunit important in muscle differentiation [Snai2 (Slug), a transcriptional repressor important in muscle differentiation [Tnrc6a (GW182), an argonaute binding partner and key factor required for all miRNA-mediated repression [To identify direct miR-30 family targets, we first utilized TargetScan 6.2  to identynthesis . From thynthesis , includiogenesis , Ppargc1n muscle , Runx1   , indicatrrelated ,30.Runx1, Smarcd2, Snai2, and Tnrc6a by miR-30a/b/c, we cloned the full length 3’-UTRs containing miR-30 target sites from C2C12 genomic DNA and inserted the fragments downstream of the Renilla luciferase coding sequence in psiCHECK-2. We then transfected these constructs into C2C12 cells along with synthetic pre-miR-30a/b/c or control pre-miR, and measured the luciferase signal following 24 hours in culture. Interestingly, we found the highest level of repression for Tnrc6a, with ∼50% and ∼60% reductions in activity for 0.5nM and 5nM pre-miR-30a/b/c, respectively (P ≤ 0.05) ∼50% reductions in luciferase activity for Smarcd2 and Snai2, known negative regulators of the myogenic gene program, indicating that miR-30a/b/c may promote myogenesis through regulation of these factors.To validate direct regulation of ectively . AdditioTnrc6a 3’-UTR was repressed ∼60% by miR-30a/b/c overexpression, we wondered if Tnrc6a repression may play a key role in regulating global miRNA-mediated repression. If miR-30 family miRNAs control miRNA repression by targeting Tnrc6a, we could expect that high levels of miR-30 would repress Tnrc6a levels resulting in global de-repression of miRNA targets and increased protein synthesis. To determine whether modulating miR-30 family miRNA levels affects miRNA repression, we tested the ability of muscle-specific miR-206 to repress a known target, cyclin D1 (Ccnd1)[Ccnd1 luciferase reporter activity (Ccnd1 repression by miR-206 (After finding that the 1 (Ccnd1), during activity , and miR miR-206 , showing3H-tyrosine incorporation assay on C2C12 cells infected with a miR-30b/d-expressing adenovirus. Indeed, after normalizing to protein content, we found a significant ∼2-fold increase (P ≤ 0.05) in 3H-tyrosine incorporation in miR-30b/d over-expressing myotubes when compared to controls , as well as by antagonizing the miRNA pathway through repression of Tnrc6a. These findings contribute to our understanding of miRNA function during myogenesis.In conclusion, we present a miRNA-seq dataset identifying a reduction in miR-30 family miRNA expression in dystrophic mdx4cv (B6Ros.Cg-Dmdmdx-4Cv/J) animals were a kind gift from Dr. Jeffrey Chamberlain (University of Washington). Animals were housed under standard conditions in a partial barrier facility and received access to water and chow ad libidum. For sample collection, animals were sedated using 1–4% inhaled isoflurane and sacrificed by cervical dislocation.All animal experiments were performed using protocols approved by the University of Colorado Institutional Animal Care and Use Committees (IACUC). C57Bl/6J animals were obtained from The Jackson Laboratory, and De-identified, archival DMD biopsy samples were kindly provided by Dr. Eric Hoffman (George Washington University) and have been described previously . Based omdx4cv mice (n = 2/group) using miRvana miRNA isolation kit (Ambion). 1 μg of small RNA per sample was then adaptor ligated, reverse transcribed and PCR amplified following a modified version of Illumina’s Small RNA Sample Prep v1.5.0. Briefly, small RNAs were first ligated to a 3’ adapter  modified with 3’-terminal dideoxy-cytosine and a 5’-adenylation, using T4 RNA Ligase 2, truncated (New England Biolabs) in the absence of ATP (to prevent RNA circularization). The RNAs were then ligated to RNA/DNA hybrid 5’ adapters, using T4 RNA Ligase 1 (New England Biolabs) in the presence of ATP. These 5’ adapters contain a three-nucleotide “barcode” sequence at their 3’ end to mark respective sample libraries. The adapter-ligated RNAs were then reverse transcribed and PCR amplified for a total of 18 cycles using primers complementary to the adapter sequences, with non-complementary overhangs containing the P5/P7 flow cell sequences. The fragments were then gel purified from an 8% polyacrylamide gel and quantified on a 2100 Bioanalyzer (Agilent). Prior to submission, libraries were validated by TOPO-cloning (Invitrogen) and conventional Sanger sequencing to ensure proper adapter orientation and insert identity. Samples were then multiplexed and submitted for single-end (36 cycle) sequencing on an Illumina GAII.To prepare the library for Illumina sequencing, small RNAs were isolated from the gastrocnemius muscles of C57Bl/6 and mdx4cv replicates were then averaged to generate expression values.Before any downstream analysis, quality assessment of the sequencing run was performed using the SolexaQA software package . Next, t-ΔΔCt method where efficiency (E) is determined using a standard curve (where perfect efficiency = 2). PCR amplification was done on a CFX96 thermocycler (Bio-Rad), with the following cycling parameters: 50°C for 2’, 95°C for 10’ followed by 40 repeated cycles of 95°C for 15” and 60°C for 1’.First, total RNA was isolated from snap frozen skeletal muscles using TRI Reagent (Molecular Research Center). miRNA expression was measured using Taqman miRNA assays (Invitrogen). Unless indicated, sno202 was used as a reference gene and 7 ng of total RNA per reaction was reverse transcribed. For mRNAs, primer sequences were chosen to amplify unique sequences and span exon-exon junctions . cDNA waBarium chloride injury was performed as described previously in Guess, et al. . Briefly2.Hindlimb suspension was performed as previously described . Briefly2C12 genomic DNA with primers  supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, 1mM sodium pyruvate and 100 U/mL penicillin/streptomycin (Life Technologies). For differentiation, 80–100% confluent cells were switched to DMEM supplemented with 2% horse serum, 2 mM L-glutamine, 1mM sodium pyruvate and 100 U/mL penicillin/streptomycin (Life Technologies). For miRNA knockdown/overexpression, 40–50% confluent cells were transfected with indicated concentrations of antimiRs (miRagen Therapeutics) or pre-miRs  using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer’s instructions. For 3’-UTR reporter transfections, 0.5 μg of indicated plasmid reporter construct was transfected along with pre-miRs into 12-well dishes in triplicate. 24 hours following transfection, cells were harvested and luciferase signals were measured using the Dual-Luciferase reporter assay kit (Promega).C2C12 cells were grown on gelatin-coated glass coverslips and transfected with 10nM pre-miRs. 24 hours following transfection, cells were fixed in 4% EM grade paraformaldehyde and stained with 1:50 anti-MYOG antibodies (Santa Cruz SC-576) and 1:200 Texas red-conjugated anti-rabbit secondary antibodies (Jackson 711-075-152) for one hour each at room temperature. For myosin heavy chain (MyHC) quantification, cells were triggered to differentiate 24 hours following transfection of 10nM antimiRs. 48 hours after triggering differentiation, cells were stained with anti-MyHC antibodies , and 1:200 FITC-conjugated anti-mouse IgG secondary antibodies (Jackson 715-095-151) for one hour at room temperature. Following antibody staining, all coverslips were then incubated in 300 nM 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature (Sigma D-9542) and mounted using Fluoromount-G (Southern Biotech). Images of the stained cells were collected using an EVOS FL fluorescence microscope, and 8–10 non-overlapping images were collected/coverslip. The images were then blindly quantified by counting total DAPI+ nuclei and either MYOG+ nuclei or total MyHC+ area using ImageJ software. Results displayed are representative of 3 independent experiments.For myogenin quantification, CFor proliferation assays, cells were electroporated with the indicated concentrations of Ambion pre-miR-control (AM17110) or pre-miR-30a-5p (PM11062) using Amaxa Nucleofector (Lonza AG) and plated on gelatin-coated glass bottom 96-well imaging plates. 24 hours following electroporation, cells were pulsed with 10 μM 5-ethynyl-2’-deoxyuridine (EdU) for 2 hours, then fixed and stained with DAPI and Alexa Fluor 555 according to the Click-iT EdU imaging kit (Life Technologies). Images were collected using an ImageXpress Micro XL (Molecular Devices) automated imaging system. Following imaging, %EdU+ cells was calculated using ImageJ software for 9 images/well for n = 4 wells.2C12 cells were grown to confluence, then switched to differentiation medium. 48 hours following the induction of differentiation, myotubes were infected with empty adenovirus (control) or adenoviruses encoding the miR-30a hairpin and the miR-30b/d cluster  at a multiplicity of infection (MOI) of 50. Adenovirus production and purification was performed as described previously [3H-tyrosine to label newly synthesized protein (CHX wells received 3H-tyrosine in addition to CHX) for 2 hours. Cellular protein was then precipitated in 10% trichloroacetic acid (TCA) for 30 minutes on ice, transferred to 1.5 mL eppendorf tubes, and spun for 5 minutes at full speed. Pellets were resuspended in 0.5 M NaOH + 0.1% Triton X-100 by incubating for 1 hour at 50°C. Scintillation counts were then measured by adding 20 uL of protein sample to 2 mL Ecoscint  + 10 uL of 1 N acetic acid. Counts were collected over one minute and normalized to total protein concentration for each sample by bicinchoninic acid (BCA) assay (Pierce).To determine rates of protein synthesis, Ceviously , using tS1 Fig(A) Alignment of miR-30a-5p, miR-30b, miR-30c, miR-30d and miR-30e shows conserved positions in bold and positions differing from miR-30a-5p in red. Seed sequence is underlined. (B) Genomic organization of miR-30 family in mice.(TIF)Click here for additional data file.S2 FigP ≤0.001 compared to miR-30a-5p, #P ≤0.05 and ## P≤0.001 compared to miR-30b. Error bars = SEM.qRT-PCR measurement of miR-30a/b/c abundance shown for WT soleus, TA, and gastrocnemius muscles relative to sno202. **(TIF)Click here for additional data file.S3 Fig2-injured muscle on indicated days post-injury (DPI). Scale bar = 200μm.H&E stained cryosections from BaCl(TIF)Click here for additional data file.S4 Fig(A) qRT-PCR measurement of miR-30a-5p in C2C12 cells transfected with indicated concentrations of pre-miR-30a-5p or pre-miR-control. **P≤0.001 compared to miR-control transfected cells at equivalent concentrations. Error bars = SEM. (B) qRT-PCR measurement of miR-30a/b/c in C2C12 cells transfected with indicated concentrations of antimiR-30 or antimiR-control. *P≤0.05, **P≤0.001 compared to antimiR-control transfected cells at equivalent concentrations. Error bars = SEM.(TIF)Click here for additional data file.S1 Tablemdx4cv and C57Bl/6 (WT) gastrocnemius muscles.Normalized expression values from miRNA-seq on whole muscle small RNAs isolated from 3-month old (PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Nasal glomangiopericytoma is rare. The immunophenotype is heterogeneous, more frequently smooth-muscle-actin and CD34-positive. We report expression patterns for several vascular-related proteins such as CD99, CD146, Bcl2, and WT1 as well as for treatment-related proteins such as mTOR and EGFR in a nasal glomangiopericytoma. The patient  presented with a left nasal tumefaction. The resected specimen (1.5-cm) showed a glomangiopericytoma. Tumor cells expressed smooth-muscle-actin, CD31, CD34, and progesterone receptor. They also expressed the vascular-cell-related proteins Bcl2, CD99, CD146, and WT1, as well as mTOR and EGFR. Nasal glomangiopericytomas show immunohistochemical heterogeneity for vascular-related markers, suggesting a possible extensive pericytic differentiation. The expression of potential targets for drug treatments such as mTOR and EGFR may impact on the clinical follow-up of these tumors occurring at advanced ages, which may require complex surgery. Nasal glomangiopericytoma is rare tumors, known to show diffuse expression of smooth-muscle-actin and focal expression of CD34. Bcl2 and CD99 are reported as negative in the few tested nasal tumors –4. Here µm thick) were stained with hematoxylin and eosin, reticulin, PAS, and reticulin. Tissue sections were also used for the immunohistochemistry techniques (Benchmark IHC/ISH automate) for the following antibodies: Bcl-2 (Dako clone 124), CD3 , CD20 (Ventana/Roche clone L-26), CD31 (Dako clone JC70A), CD34 (Dako clone QBEnd-10), CD56 (Novocastra clone 1B6), CD99 (Dako clone 12E7), CD117 , CD133 , chromogranin (Dako clone DAK-A3), desmin (Dako clone D33), EMA (Dako clone E29), estrogen receptor (Novocastra clone 6F11), HER2 (Ventana/Roche clone 4B5), HMB45 (Dako clone HMB45), Ki67 (Ventana/Roche clone 4A4), melan A (Dako clone A103), mTOR , progesterone receptor (Novocastra clone 16), Ros1 , smooth-muscle-actin , synaptophysin (Ventana/Roche clone SP11), S100 protein , and WT1 (Cell Marque clone 6F-H2). For the anti-CD146 antibody , the immunohistochemistry technique was performed manually [The patient  complained of nasal tumefaction. The medical history included left colon cancer (32 years previously) and arterial hypertension. On rhinoscopy there was a left nasal, septal, polypoid lesion. This lesion was resected. The specimen was fixed in formalin 10% and then included in paraffin. Tissue sections (3 Here we report immunohistochemical heterogeneity for vascular-related markers in a case of nasal glomangiopericytoma. The immunophenotype of tumor cells in the present case consisting in Bcl2, CD99, and progesterone-receptor positivity and with very focal expression of smooth-muscle-actin, CD31, and CD34 was suggestive for a meningeal hemangiopericytoma rather than for an extracranial hemangiopericytoma/solitary fibrous tumor-type lesion . However