The DEAD box helicase DDX3 assembles IPS-1  in non-infected human cells where minimal amounts of the RIG-I-like receptor (RLR) protein are expressed. DDX3 C-terminal regions directly bind the IPS-1 CARD-like domain as well as the N-terminal hepatitis C virus (HCV) core protein. DDX3 physically binds viral RNA to form IPS-1-containing spots, that are visible by confocal microscopy. HCV polyU/UC induced IPS-1-mediated interferon (IFN)-beta promoter activation, which was augmented by co-transfected DDX3. DDX3 spots localized near the lipid droplets (LDs) where HCV particles were generated. Here, we report that HCV core protein interferes with DDX3-enhanced IPS-1 signaling in HEK293 cells and in hepatocyte Oc cells. Unlike the DEAD box helicases RIG-I and MDA5, DDX3 was constitutively expressed and colocalized with IPS-1 around mitochondria. In hepatocytes (O cells) with the HCV replicon, however, DDX3/IPS-1-enhanced IFN-beta-induction was largely abrogated even when DDX3 was co-expressed. DDX3 spots barely merged with IPS-1, and partly assembled in the HCV core protein located near the LD in O cells, though in some O cells IPS-1 was diminished or disseminated apart from mitochondria. Expression of DDX3 in replicon-negative or core-less replicon-positive cells failed to cause complex formation or LD association. HCV core protein and DDX3 partially colocalized only in replicon-expressing cells. Since the HCV core protein has been reported to promote HCV replication through binding to DDX3, the core protein appears to switch DDX3 from an IFN-inducing mode to an HCV-replication mode. The results enable us to conclude that HCV infection is promoted by modulating the dual function of DDX3. The retinoic acid inducible gene-I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) encode cytoplasmic RNA helicases DDX3 acts on the IFN-inducing pathway by a complex mechanism. Early studies reported that DDX3 up-regulates IFN-beta induction by interacting with IKKepsilon DDX3 resides in both the nucleus and the cytoplasm A number of reports have demonstrated the formation of the DDX3-core protein complex in the cytoplasm, but the functional relevance of DDX3-core protein interaction is not known. In this report, we show evidence that the HCV core protein participates in suppression of DDX3-augmented IPS-1 signaling for IFN-β induction. Several possible functions of DDX3 are discussed, focusing on its core protein association and IPS-1-regulatory properties.HEK293 cells and HEK293FT cells were maintained in Dulbecco's Modified Eagle's low or high glucose medium  supplemented with 10% heat-inactivated FCS (Invitrogen) and antibiotics. Huh7.5 cells were maintained in MEM  supplemented with 10% heat-inactivated FCS. Hepatocyte sublines with HCV replicon (O cells) and without replicon (Oc cells) were established as described previously  GAG CCA CCA TGA GTC ATG TGG CAG TGG AA) and DDX3C R-Ba (GGA TCC GTT ACC CCA CCA GTC AAC CCC) from human lung cDNA library. To make an expression plasmid, HA tag was fused at the C-terminal end of the full length DDX3 (pEF-BOS DDX3-HA). pEF-BOS DDX3 (1-224aa) vector was made by using primers, DDX3 N-F-Xh and DDX3D1 (GGA TCC GGC ACA AGC CAT CAA GTC TCT TTT C). pEF-BOS DDX3-HA (225-662) was made by using primers, DDX3D2-3 (CTC GAG CCA CCA TGC AAA CAG GGT CTG GAA AAA C) and DDX3C R-Ba. To make pEF-BOS DDX3-HA (225-484) and pEF-BOS DDX3-HA (485-663), the primers, DDX3D2 R-Ba (GGA TCC AAG GGC CTC TTC TCT ATC CCT C) and DDX3D3 F-Xh (CTC GAG CCA CCA TGC ACC AGT TCC GCT CAG GAA AAA G) were used, respectively. HCV core expressing plasmids, pcDNA3.1 HCVO core or JFH1 core, were previously reported by N. Kato (Okayama University Japan) DDX3 cDNA encoding the entire ORF was cloned into pCR-blunt vector using primers, DDX3N F-Xh . cDNA of HCV 3′ UTR region was amplified from total RNA of O cells using primers HCV-F1 and HCV-R1, and then cloned into pGEM-T easy vector. The primer set sequences were HCV-F1: CTC CAG GTG AGA TCA ATA GG and HCV-R1: CGT GAC TAG GGC TAA GAT GG. RNA was synthesized using T7 and SP6 RNA polymerases. Template DNA was digested by DNase I, and RNA was purified using TRIZOL (Invitrogen) according to manufacturer's instructions.The HCV genotype 1b polyU/UC RNA  5′-GAU UCG UAG AAU AGU CGA ACA-3′, siRNA-2: 5′-GGA GUG AUU ACG AUG GCA UUG-3′, siRNA-3: 5′-GCC UCA GAU UCG UAG AAU AGU-3′ and control siRNA: 5′-GGG AAG AUC GGG UUA GAC UUC-3′. 20 pmol of each siRNA was transfected into HEK293 cells in 24-well plate with Lipofectamin 2000 according to manufacture's protocol. Knockdown of DDX3 was confirmed 48 hrs after siRNA transfection. Experiments were repeated twice for confirmation of the results.Knockdown of DDX3 was carried out using siRNA, DDX3 siRNA-1: 4 cells/well) cultured in 24-well plates were transfected with the expression vectors for IPS-1, DDX3 or empty vector together with the reporter plasmid (100 ng/well) and an internal control vector, phRL-TK (Promega) (2.5 ng/well) using FuGENE (Roche) as described previously Firefly and Renella luciferase activities were determined using a dual-luciferase reporter assay kit (Promega). The Firefly luciferase activity was normalized by Renella luciferase activity and is expressed as the fold stimulation relative to the activity in vector-transfected cells. Experiments were performed three times in duplicate (otherwise indicated in the legends).HEK293 cells  (Sigma) in the culture medium, and the cell culture supernatant was replaced with the medium containing polyI:C and DEAE-dextran. Using DEAE-dextran, polyI:C is incorporated into the cytoplasm to activate RIG-I/MDA5.in vitro by T7 RNA polymerase, transfected into HEK293 cells in 24-well plate by lipofectamin 2000 (Invitrogen) with other plasmids. Cells were allowed to stand for 24∼48 hrs and HCV RNA-enhancing activation of IFN-beta promoter was assessed by reporter assay.HCV 3′ UTR poly U/UC region (PU/UC) RNA (0∼50 ng/well), which is synthesized 3Vo4, 20 mM IAA and 2 mM PMSF), and the protein was immunoprecipitated with anti-HA polyclonal (SIGMA) or anti-FLAG M2 monoclonal Ab (SIGMA). The precipitated samples were resolved on SDS-PAGE, blotted onto a nitrocellulose sheet and stained with anti-HA (HA1.1) monoclonal (SIGMA), anti-HA polyclonal or anti-FLAG M2 monoclonal Ab.HEK293FT cells were transfected in a 6-well plate with plasmids encoding DDX3, IPS-1, RIG-I or MDA5 as indicated in the figures. 24 hrs after tranfection, the total cell lysate was prepared by lysis buffer CAC UUU CAC UUC UCC CUU UCA GUUU. The biotin is conjugated at U residue at the 3′ end of antisence strand (underlined). Biotinylated double-stranded (ds)RNA were incubated for 1 hr at 25°C with 10 µg of protein from the cytoplasmic fraction of cells that were transfected with Flag-tagged RIG-I and HA-tagged DDX3 expressing vectors. The mixture was transferred into 400 µl of lysis buffer containing 25 µl of streptavidine Sepharose beads, rocked at 4°C for 2 h, collected by centrifugation, washed three times, resuspended in SDS sample buffer.The pull-down assay was performed according to the method described in Saito T et al. RNA-binding proteins were identified by affinity chromatography and Mass spectrometry. Briefly, cell lysate was prepared from human HEK293 or Raji cells as will be described elsewhere . The lysate was first applied to polyU-Sepharose and then the pass-through fraction was applied to PolyI:C-Sepharose. The eluted proteins were analyzed on Mass spectrometry using the MASCOT software.HCV replicon-positive (O) or –negative (Oc) cells were plated onto cover glass in a 24-well plate. In the following day, cells were transfected with indicated plasmids using Fugene HD (Roch). The amount of DNA was kept constant by adding empty vector. After 24 hrs, cells were fixed with 3% of paraformaldehyde in PBS for 30 minutes, and then permeabilized with PBS containing 0.2% of Triton X-100 for 15 min. Permeabilized cells were blocked with PBS containing 1% BSA, and were labeled with anti-Flag M2 mAb (Sigma) or anti-HA pAb (Sigma) in 1% BSA/PBS for 1 hr at room temperature We have performed proteome analyses of RNA-binding fractions in human dendritic cell lysate eluted from polyU and polyI:C Sepharose. 127 cytoplasmic proteins were reproducibly identified as polyI:C-binding proteins . Four of them are DEAD/H box helicases. In this setting, we found DDX3 is a RNA-binding protein . DDX3 inUsing biotinylayed dsRNA, RNA-binding properties of DDX3 and RIG-I were tested by pull-down assay. DDX3 or RIG-I protein was co-precipitated with dsRNA in HEK293 cells expressing either alone of DDX3 or RIG-I . StrikinA polyU/UC sequence is present in the 3′-region of the HCV genome, and serves as a ligand for RIG-I in IPS-1 pathway activation DDX3 mRNA  and protWe next determined whether the HCV replicon triggers IPS-1/DDX3 IFN promoter activation, using human hepatocyte lines with the HCV replicon (O cells) or without it (Oc cells). In O cells with the HCV replicon, IPS-1/DDX3 expression showed minimal enhancement of IFN-beta promoter activation , while iWhat HCV proteins participate in IFN-beta induction was tested in a pilot study using protein expression analysis. We found that expression of HCV core protein as well as NS3/4A led to suppression of IFN-beta reporter activity in Oc cells (data not shown). The HCV core protein physically binds DDX3 Although some endogenous DDX3 was present in the cytoplasm without DDX3 transfection, only IPS-1 transfection permitted minimal induction of IFN-beta. It is notable that high doses of the HCV JFH1 core protein was needed to inhibit the IPS-1-mediated IFN-beta-induction signal . Since tIPS-1 transduces a RNA replication signal to result in IFN-beta output using downstream proteins, such as NAP1 and IKKeysilon. If the HCV core protein interferes with IPS-1 function through DDX3, the core should not inhibit over-expressed downstream molecules. As predicted, HCV core protein did not suppress the IKKepsilon- or NAP1-mediated IFN-beta-inducing signal . An upstTo further confirm this model, we examined whether the HCV core protein inhibits the physical interaction between IPS-1 and DDX3. Full length IPS-1 and the C-terminal fragment of DDX3, which binds to the IPS-1 CARD-like region, were transfected into HEK293 cells, with or without the HCV core protein, and the DDX3 fragment was immunoprecipitated. Expression of HCV core proteins strongly inhibited interaction between the DDX3 C-terminal fragment and IPS-1 . JFH1 coPolyU/UC HCV RNA activates the IFN-beta promoter , and thiWe attempted to confirm this finding by tag-expressed proteins and imaging analysis. In Huh7.5 cells IPS-1 colocalized with DDX3 around the mitochondria , and so What happens in the O cells with replicon when the core protein is expressed was next tested. Using O and Oc cells, we tested the localization of the core protein and DDX3 in comparison with IFN-inducing properties . In O ceOverexpressed DDX3 allowed the Oc cells to induce IPS-1-mediated IFN-beta promoter activation , while tWe investigated the effect of the HCV core protein on the cytosolic DDX3 that forms a complex with IPS-1 to enhance the RIG-I-mediated RNA-sensing pathway. We demonstrated that the core protein removes DDX3 from the IFN-β-inducing complex, leading to suppression of IFN-β induction. DDX3 is functionally complex, since its protective role against viruses may be modulated by the synthesis of viral proteins. DDX3 acts on multiple steps in the IFN-inducing pathway It remains unclear in Together with these findings, the results presented here suggest that the HCV core inactivates IPS-1 in a mode different from NS3/4A DDX3 appears to be a prime target for viral manipulation, since at least three different viruses, including HCV A continuing question is the physiological role of the molecular complex of DDX3 and IPS-1 during replication of HCV in hepatocytes. HCV proteins generated in host hepatocytes usually induce an HCV-permissive state in patients, for example in the IFN-inducing pathways. NS3/4A protease induces rapid degradation of IPS-1 DDX3 primarily is an accelerating factor for antiviral response through IPS-1-binding. Many host proteins other than DDX3 may positively regulate HCV replication in hepatocytes in association with the IPS-1 pathway. In this context, we know LGP2 Imaging studies suggest that the complex of IPS-1 involving the membrane of mitochondrial/peroxisomes differ from that free from the membrane. Although IPS-1 is liberated from the membrane by NS3/4A having largely intact cytosolic domain, it loses the IFN-inducing function Evidence is accumulating that HCV checks many steps in the IFN-inducing pathway throughout the early and late infection stages, and suppresses IFN production by multiple means. Disruption of IPS-1 function by both NS3/4A and core protein may be crucial in HCV-infected Huh7.5 cells, even though the cells harbor dysfunctional RIG-I Figure S1The IPS-1 complex. IPS-1 and HCV core bind C-terminal regions of DDX3. DDX3 captures dsRNA at the C-terminal domain. This figure is constructed from (0.41 MB TIF)Click here for additional data file.Figure S2DDX3 enhances RIG-I-mediated IFN-β promoter activation induced by polyI:C. (A) DDX3 si-1 or control siRNA was transfected into HEK293 cells with reporter plasmids and RIG-I-expression plasmid or control plasmid (100 ng). After 48 hrs, cells were stimulated with polyI:C (20 µg/ml) with dextran for 4 hrs, and activation of the reporter p125luc was measured. (B) MDA5 (25 ng), IPS-1 (100 ng), DDX3 (100 ng), JFH1 core (50 ng) and/or p125 luc reporter (100 ng) plasmids were transfected with HEK293 cells. Cell lysates were prepared after 24 hrs, and luciferase activities measured. The results are representative of two independent experiments, each performed in triplicate.(0.17 MB TIF)Click here for additional data file.Figure S3DDX3 colocalizes with IPS-1 on the mitochondria in Huh7.5.1 cells. HA-tagged DDX3 and FLAG-tagged IPS-1 were co-transfected into Huh7.5.1 cells. After 24 hrs, cells were fixed with formaldehyde and stained with anti-HA polyclonal and FLAG monoclonal Abs. Alexa488 (DDX3-HA) or Alexa633 antibody was used for second antibody. Mitochondria were stained with Mitotracker Red. A representative result from three independent experiments is shown.(0.92 MB TIF)Click here for additional data file.


It suggested thatcopper aspirinate possesses potential neuroprotective properties, the mechanism of which might be related toan increase of the activity of endogenous superoxide dismutase.The cerebroprotective effects of copper aspirinate [dimeric copper(II) bis(o-acetoxybenzoate)] wereinvestigated in gerbils subjected to 10-min global cerebral ischemia followed b 60-min reperfusion. Theresults showed that intragastric copper aspirinate (7.5, 15.0 and 30.0 mg Kg


Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2δ assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC50-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves  The channels , 4, repochannels , 6 or Bachannels , 8 have channels -12.Recent advances in automated uHTS technologies have enabled the quantitative and parallel interrogation of arrays of cellular assays , 14. In In vitro, a minimal reagent set of three peptide substrates in conjunction with one phospho-specific antibody [TM-platform, Cisbio International). We have translated this biochemical platform into a cellular format while maintaining the original homogeneous addition-only protocol as well as the HTS-friendly TR-FRET readout [Here, we present the foundation of a cellular Ser/Thr-kinase assay platform that relies on promiscuous peptide substrates for the quantification of intracellular kinase activities. antibody  is suffi readout -21. In tEuropium-conjugated pSTK (61PSTKAY), pAkt (61P15 KAYC) pMEK1/2 (61P17KAYC), cMyc (61MYCKLB) antibodies and XL665-conjugated FLAG (61FG2XLB) and His6 (61HISXLB) antibodies were purchased from Cisbio, US. Unconjugated B-Raf antibody was from Santa Cruz Biotechnology (sc-5284), pMEK1/2 antibody from Cell Signaling Technology (9121), anti-FLAG M2 from Sigma. TR-FRET assays were performed in white tissue culture 384-well or 1536-well plates (Greiner Bio One). pcDNA5TO plasmid, HEK293 T-Rex cells, hygromycin, zeocin, and blasticidin were purchased from Invitrogen. Plasmid pSEMS1-26m was obtained from Covalys. Wortmannin and LY294002 were from Calbiochem and staurosporine from Sigma. Other reference compounds were synthesized at GNF.i.e. the SNAP-tag [Peptide substrate sequences were epitope-tagged and fused to AGT, SNAP-tag -24. GeneSNAP-tag . For simFull-length B-Raf wild-type (WT) and B-Raf(V600E) (NM_004333) cDNAs were subcloned from pcDNA3.1 into pcDNA5TO (Invitrogen) using HindIII/ Not1 restriction enzyme sites. The kinase-inactive (KD) double mutant B-Raf  was generated by site-directed mutagenesis. Akt1 (NM_005163) cDNA was subcloned from pcDNA3.1 into pcDNA5TO (Invitrogen) using BamHI/XbaI restriction enzyme sites and a C-terminal His-tag was added to the amino-terminus by PCR. The kinase-inactive AKT-KD (K179M) point mutant was generated by site-directed mutagenesis. HEK293 T-Rex cells (Invitrogen) were cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone) and 5μg/ml blasticidin. Stable pools of cells constitutively expressing either cSTKpeptide or MEK1 protein substrates were generated by transfection of cDNA plasmids and selection in G418 (1 mg/ml) or zeocin (200 μg/ml), respectively. Double stable cell lines expressing tetracycline-inducible kinases were subsequently generated by transfection of these “substrate-only” HEK293 T-Rex cells and selection in medium containing 175μg/ml hygromycin B.HEK293 cells were transfected using Fugene 6 (Roche) at a density of 8000 cells/well in 384-well plates and a final transfection volume of 30μl. The amounts of kinase and substrate plasmids per well varied for each experiment and are specified in the figure legends. Cell lysis and detection of phosphorylated or total substrate levels was performed 48h post transfection by addition of 30μl detection buffer  containing anti-FLAG XL665 (2ng/ul) and Eu-pSTK (0.18ng/μl) or Eu-pMEK1 (0.24ng/μl). For the relative determination of total substrate levels 30μl detection buffer was added per well containing anti-FLAG XL665 (2ng/μl) and Eu-cMyc (0.3ng/μl). Plates were incubated for 3-6h at room temperature and TR-FRET readings taken on Acquest, Analyst GT (Molecular Devices Corp.), Envision or Viewlux (Perkin Elmer) plate readers. Emission readings at 671nm and 618nm were expressed as a ratio and multiplied by a constant value of 100 to obtain “TR-FRET Ratios”.cDNA libraries and screening protocols were applied essentially as previously described , 26. BriFor LMW screening purposes in 384-well plates HEK293 T-Rex cells were either transiently transfected with tetracycline-inducible kinase and substrate constructs at a previously optimized ratio following the above mentioned reverse transfection protocol. Alternatively, double stable cells were plated directly at a density of 8,000cells/well in a volume of 30μl. 24h post plating, tetracycline (1μg/ml) was added to induce kinase expression for approximately 16h prior to compound addition. Kinase inhibitors were resuspended in DMSO and 12 point 1:3 serial dilutions were prepared. 100nl of compound stocks were pintool transferred into the assay plates resulting in a final DMSO concentration of 0.3%. After a 2h incubation period TR-FRET detection reagents detection reagents were added. For 1536-well plate screening cells were either transiently transfected in bulk or stable cells were seeded directly. For transient bulk transfections the transfection reagent mixture was prepared according to the following ratios: plasmid DNA: fugene 6: serum Free media = 1μg: 3μl: 21μl. For every 1ml of cells 1.25μg of kinase plasmid, 0.156μg of substrate plasmid (8:1), 4.22μl of Fugene 6 and 30μl of serum free media was prepared. The transfection mixture was then incubated at room temperature for 30 min and mixed into a HEK293 cell suspension (400 cells/μl). 2,000 cells/well were dispensed in 1536-well plates in 5μl media volumes and incubated at 37˚C. Kinase expression was induced in stable cells after 24h incubation through the addition of 1μl tetracycline (5μg/ml) followed by an additional 4-16 h incubation time before compound addition. Compound treatment in transient transfection experiments occurred 48h post seeding. In either case, 50nl DMSO stocks were pintool transferred into each well and incubated at 37˚C for a 2h period before addition of 4μl detection reagents. TR-FRET readings were taken after another incubation time of 4-24h. Z’ scores were determined as described .2EDTA, 1 mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1 mM Beta-glycerophosphate, 1 mM Na3VO4, 1ug/ml leupeptin, supplemented with 1X protease inhibitors (Sigma). Cellular extracts were resolved by SDS-PAGE , transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. Immunolabeled proteins were visualized through Odyssey infrared imaging , with IRDyeTM800  and AlexaFluor® 680 (Molecular Probes)-conjugated fluorescent secondary antibodies.HEK293 cells were harvested 48h post transfection in cell lysis buffer -Ba/F3 cell line was generously provided by Markus Warmuth (GNF). Ba/F3 cells were counted and diluted to a final concentration of 75,000 cells/ml in RPMI media and 50μl/well were plated into 384-well white solid bottom plates. 50nl of a 10mM compound stock in DMSO were pintool transferred followed by a 48h incubation time at 37°C for 48h. Finally, 25μl Cell-Titer-Glo (Promega) detection reagent was added each and luminescence read were taken on a CLIPR (Molecular Devices Corp.) reader.1A, [In vitro, the incubation of purified kinases with biotinylated peptide substrates leads to substrate phosphorylation quantifiable by TR-FRET [Cisbio’s KinEASE platform utilizes a minimal reagent set of three promiscuous peptide substrates (STK-peptides) sharing a common Ser-phosphorylation site and one corresponding phospho-specific antibody (pSTK) to support a panel of >100 biochemical Ser/Thr-kinase assays . In vit TR-FRET . We soug1B). The added epitope tags enable the quantification of either total substrate expression or substrate phosphorylation. To have a reference for peptide based cellular Ser/Thr-kinase assays we developed MEK1 as a model full-length protein substrate of kinases such as B-Raf or B-Raf(V600E), an activated oncogenic kinase mutant and established cancer drug target [To this end, we flanked the STK peptide sequences with N-terminal Myc- and C-terminal Flag-epitopes. The resulting oligopeptides were fused to hAGT carrier proteins (“SNAP-tag”) -24 to sug target , 32.2A, C and E). Similar observations were made with peptide substrates cSTKpep-1 and cSTKpep-2 .When the cSTKpep-3 substrate was expressed alone in HEK293 cells the observed basal phosphorylation levels derived from endogenous Ser/Thr-kinase activities were low, barely detectable by TR-FRET with weak phospho-specific bands being detectable by western blotting . Moreover, B-Raf(V600E)-induced cSTKpep-3 phosphorylation was both dependent on the kinase activity as well as the presence of the target phosphorylation site since no cSTKpep-3 phosphorylation was detected when using a kinase-inactive mutant version of B-Raf(V600E), B-Raf(V600E)-KD or a Ser/Ala-mutant of cSTKpep-3 (cSTK pep-3-SA) .Cotransfection of cSTKpep-3 with B-Raf(V600E) resulted in a several fold increase in substrate phosphorylation by both western blotting and TR-FRET . Comparable results were obtained when using kinase-inactive, MEK1(KD), as a substrate ; cotransfection of MEK1 with B-Raf(V600E) resulted in increased MEK1 phosphorylation as detected by both western blotting and TR-FRET.As expected, cSTKpep-3 phosphorylation could be modulated by both the amounts of substrate and kinase plasmids transfected as well as by the activation state of the transfected kinase .For the pharmacological evaluation we generated double stable cells constitutively expressing either cSTKpep-3, wild-type MEK1(WT) or kinase-inactive MEK1(KD) substrates in combination with Tet-inducible B-Raf(V600E). Kinase inhibitors were then tested in 384-well or 1536-well formats. In all cases short compound treatment times of around 2h were employed to minimize cytotoxicity related compound interference . Good agreement was also observed when comparing TR-FRET assay results with historical Ba/F3-proliferation data for B-Raf(V600E) . None of these B-Raf inhibitors affected basal cSTKpep-3 and MEK1(WT) background phosphorylation in the absence of prior B-Raf(V600E) induction (data not shown). Finally, a selection of unrelated kinase inhibitors, including Wortmannin and LY29002 did not show any activity in either the cSTKpep-3 or MEK1(WT) TR-FRET assays (Table 1).Overall, cellular TR-FRET assays performed very robustly with B-Raf inhibitor A in both 384-well and 1536-well plates for MEK(WT), MEK(KD) or c-STKpep-3 substrates with Z’=0.6-0.9. Moreover, the observed potencies of a panel of selective in-house B-Raf inhibitors correlated very well in cSTKpep-3 and MEK1(WT) TR-FRET-assays (R1), pronounced discrepancies were reproducibly observed for the activities of the highly selective MEK1/2 inhibitor PD0325901 [in vitro. Both compounds had comparable activities when using cSTKpep-3 or MEK1(WT) as B-Raf(V600E) substrates, but were inactive when using MEK1(KD). Similar observations of substrate dependent inhibitor profiles in cellular MEK-phosphorylation assays were recently made using an ELISA readout [While B-Raf selective compounds compared very well in the above assays . In the absence of other efficacious AKT inhibitors staurosporine was used as a tool compound and found to effectively inhibit AKT-dependent cSTKpep-3 phosphorylation . Its cellular potency (IC50 ~100nM) agreed with the in vitro activity against AKT (IC50~100nM). Comparable IC50 values were found for incubation periods ranging from 30min (IC50~240nM) to 2h (IC50~100nM) (data not shown).For a second case study we turned to AKT and its upstream activation pathway. AKT-dependent intracellular cSTKpep-3 phosphorylation could be detected by western blotting and by TR-FRET . After short 2h treatment staurosporine exclusively affected cSTKpep-3 phosphorylation, but not cSTKpep-3 expression levels. After an extended 48h treatment stauros-porine affected phospho- as well as total-cSTKpep-3 levels with equal apparent potency, suggesting that the occurrence of cytotoxicity inappropriately interfered with the assay readout.To determine if staurosporine-induced cytotoxicity and the subsequent loss of kinase substrate expression affected the outcome of the cellular assays, we aimed to separate the effect of staurosporine on cSTKpep-3 phosphorylation from its effect on total cSTKpep-3 expression . To this end we developed another TR-FRET assay using His-tagged AKT to monitor Ser473 phosphorylation.Next, we aimed to interrogate the AKT pathway in greater detail by separating AKT activation as indicated by Ser473 phosphorylation, from signaling events depending on activated AKT such as cSTKpep-3 phosphorylation  and cSTKpep-3 phosphorylation  assays. Moreover, the observed potencies of Wortmannin and LY294002 in the low nanomolar and low micromolar concentration ranges respectively were in line with their reported in vitro and cellular activities on PI3K [50~6μM) versus p-cSTKpep-3 (IC50~60nM). These cellular activities mirror the in vitro potency of staurosporine on PI3K (IC50~9μM) and AKT (IC50~100nM).As predicted for inhibitors of AKT activation, the PI3K inhibitors LY294002 and Wortmannin displayed comparable potencies in both AKT phosporylation . Of these 22 Ser/Thr-kinases, 13 were already known members of the biochemical KinEASE-panel, whereas 9 kinases were newly identified. Nine out of the 13 previously known kinases preferred the same STK peptide substrate in vitro and in cells (Table 2). No Tyr-kinases were found to stimulate cSTKpep phosphorylation, suggesting that no indirect Tyr-kinase induced activation of endogenous Ser/Thr-kinase pathways triggered cSTKpep phosphorylation. Overall, these observations are consistent with the idea that the intracellular kinase-cSTKpep interactions were direct.Having shown proof-of-concept for two important drug targets AKT and B-Raf, we sought to identify additional Ser/Thr-kinases amenable to our cellular assay approach through cDNA screening. A kinase focused sublibrary of GNF’s genome scale cDNA collection , 26 compvia protein engineering, e.g. through the deletion of autoinhibitory domains or activating mutations, may be required beyond their plain overexpression to induce cSTKpep phosphorylation. The subcellular localization of kinases or their substrates, cell cycle dependency, cellular backgrounds and the dynamics of signaling pathways all may play a role. By rescreening our kinase cDNA library under modified assay conditions, e.g. in the presence of external stimuli, nuclear targeted substrates, different cell types, or alternatively through the design of an entirely new cDNA library of activated kinase constructs we aim to further expand the current panel of cellular Ser/Thr-kinase assays in the future. In the case of CamK2δ we found that transient activation of kinase activity and subsequent cSTKpep-1 phosphorylation was ionomycin inducible . We believe that assay conditions can be found to activate additional Ser/Thr-kinases in a similar manner.While a number of kinases already represented on the biochemical KinEASE panel were picked up through DNA screening, others were missed. Several reasons might help explain these findings. Preactivation of Ser/Thr-kinases either through physiologic stimuli or 50 values. This specific feature of the presented cellular TR-FRET kinase platform tremendously benefits HTS applications as it avoids artifactual cytotoxic hits or non-specific transcriptional inhibitors which tend to interfere with traditional proliferation or reporter gene assays of Ser/Thr-kinase activity. The builtin option to quantify relative substrate amounts as well as substrate phosphorylation by TR-FRET and thereby to normalize for substrate expression levels further helps to sort out non-specific compound interference. Finally, having validated cellular TR-FRET assays for two important drug targets, we expanded our assay concept to seed a panel of currently >20 cellular Ser/Thr-kinase assays through cDNA screening. While this initial panel will mainly target full-length wild-type kinases which are constitutively activated upon overexpression in HEK293 cells, a future generation of assays can be envisioned to include also engineered Ser/Thr-kinases or to involve kinase activation through physiologic stimuli. Wherever high-quality phospho-specific antibodies are available, protein kinase substrates will be incorporated into our panel as well. However, due to the very limited availability of such reagents it is expected that a rapid expansion and a significant coverage of the Ser/Thr-kinome will be initially achieved through TR-FRET assays relying on more broadly applicable peptide substrates like cSTKpep-1, 2 and -3 in conjunction with the corresponding universal p-STK antibody [Here we present a starting point for the construction of a new cellular Ser/Thr-kinase assay platform that is tailored towards automated high density compound profiling and primary uHTS. We demonstrated that generic peptide substrates can form a viable basis for the determination of intracellular Ser/Thr-kinase activities and that a homogeneous TR-FRET protocol can be formulated to detect peptide phosphorylation in a sensitive, quantitative and automation-friendly manner. For B-Raf we showed that peptide- and protein-based cellular TR-FRET assays returned well correlated activity profiles for a panel of tool compounds ranging from highly selective to relatively promiscuous kinase inhibitors. Despite the overexpression of target kinases high-potency inhibitors could be readily identified as such. In addition, overexpressed target kinases like AKT and CamK2δ remained sensitive to upstream activating signals. Importantly, short compound incubation times on the order of 1-2 hours were sufficient to obtain robust ICantibody , 21. In


C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase II genes and found that nuc-1 plays a major role, crn-6 plays an auxiliary role, and crn-7 plays a negligible role in resolving 3′ OH DNA breaks generated in apoptotic cells. Promoter swapping experiments suggest that crn-6 but not crn-7 can partially substitute for nuc-1 in mediating apoptotic DNA degradation and both fail to replace nuc-1 in degrading bacterial DNA in intestine. Despite of their restricted and largely non-overlapping expression patterns, both CRN-6 and NUC-1 can mediate apoptotic DNA degradation in many cells, suggesting that they are likely secreted nucleases that are retaken up by other cells to exert DNA degradation functions. Removal or disruption of NUC-1 secretion signal eliminates NUC-1's ability to mediate DNA degradation across its expression border. Furthermore, blocking cell corpse engulfment does not affect apoptotic DNA degradation mediated by nuc-1, suggesting that NUC-1 acts in apoptotic cells rather than in phagocytes to resolve 3′ OH DNA breaks. Our study illustrates how multiple DNase II nucleases play differential roles in apoptotic DNA degradation and development and reveals an unexpected mode of DNase II action in mediating DNA degradation.DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event.  Programmed cell death, or apoptosis, is a conserved cellular process critical for animal development and tissue homeostasis. During apoptosis, a series of morphological changes occur due to the activation of the cell killing caspases, including chromatin condensation and fragmentation.2+ for optimal activity to generate DNA fragments with 3′ hydroxyl ends (3′OH) Multiple nucleases have been identified to catalyze fragmentation of chromosomal DNA during apoptosis. DFF40 (40 kDa DNA fragmentation factor) or CAD (caspase-activated DNase) plays a major role in generating internucleosomal DNA ladders during mammalian apoptosis 2+/Mg2+-independent and has maximum activity at acidic conditions (pH 5.0–6.0) In contrast to DFF40/CAD, DNase II is CaC. elegans, no DFF40/CAD or DFF45/ICAD homologues have been identified nuc-1 and cps-6 loss-of-function (lf) mutants, increased numbers of apoptotic cells are stained by TUNEL, which specifically labels 3′ OH DNA breaks, suggesting that NUC-1 and CPS-6 play a role in resolving DNA fragments with 3′ OH ends in apoptotic cells. Interestingly, loss of cps-6 delays progression of apoptosis and can even block cell death in sensitized genetic backgrounds nuc-1 does not seem to affect cell killing or the kinetics of cell death nuc-1 and cps-6 play different roles in apoptosis, with cps-6 acting early during apoptosis and nuc-1 functioning at a later stage of apoptosis. Moreover, in an RNAi-based functional genomic screen, seven additional cell death-related nucleases (CRNs) have been identified In C. elegans, CRN-6 (K04H4.6) and CRN-7 (F09G8.2). crn-6(RNAi) causes increased number of TUNEL-positive cells in wild-type C. elegans embryos as well as in nuc-1(lf) embryos, suggesting that CRN-6 acts in parallel to NUC-1 to promote apoptotic DNA degradation cps-6 and other crn genes, crn-6 does not affect cell killing or the kinetics of apoptosis and may act at a later stage of apoptosis like nuc-1crn-7 in apoptotic DNA degradation is unclear. In addition to its role in apoptosis, nuc-1 plays a role in degrading DNA of ingested bacteria in the intestinal lumen In addition to NUC-1, there are two other DNase II homologues in C. elegans apoptosis and development, we analyze their expression patterns, their mutant phenotypes, their activities in mediating DNA degradation in promoter swapping experiments, and the importance of their secretion to their DNA degradation functions. Our results indicate that three C. elegans DNase II genes play differential roles in C. elegans apoptosis and development and provide unique mechanistic insights into how DNase II nucleases mediate apoptotic DNA degradation.To investigate the roles of three DNase II genes in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique nuc-1(lf) mutants as well as in crn-6(RNAi) animals Chromosomal DNA fragments generated during apoptosis often contain 3′ OH ends that can be labeled by the C. elegans DNase II homolgues, we analyzed the loss-of-function phenotypes of these genes. We isolated an 843 bp deletion mutation (tm890) in crn-6 that removes exons 3–6 of crn-6 and is likely a null allele. We also obtained a 527 bp deletion (ok866) in crn-7 that removes one third of exon 2 and almost all intron 2 , crn-6(tm890) and crn-7(ok866) mutant embryos all had more TUNEL-positive signals than N2 embryos (nuc-1(e1392) mutant embryos had approximately 30 TUNEL-positive signals at these three embryonic stages (nuc-1 mutant embryos must be DNA breaks that were generated during early embryonic stages and persisted through the late embryonic stages crn-6(tm890) mutant embryos had an average 10.2, 11.0 and 0.7 TUNEL-positive signals, whereas crn-7(ok866) mutant embryos had only 6.0, 6.9 and 0.4 TUNEL-positive nuclei, at the comma, 1.5-fold, and 4-fold embryonic stages, respectively. The increased TUNEL-positive signals observed in crn-6 and crn-7 mutant embryos were completely abolished by a ced-3(n2433lf) mutation , crn-6(tm890), and crn-7(ok866) single mutant, and the crn-7 crn-6; nuc-1 triple mutant ; nuc-1(e1392) double mutant exhibited a modest increase in TUNEL- positive signals in 1.5-fold embryos but contained 58% more TUNEL-positive cells  in 4-fold embryos than nuc-1(e1392) 4-fold embryos  4-fold embryos had an average of 0.7 TUNEL signal. This observation suggests that CRN-6 plays an auxiliary role to NUC-1 in degrading chromosomal DNA during apoptosis, especially in late embryonic stages. No obvious difference in TUNEL staining was seen between nuc-1(e1392) and crn-7(ok866); nuc-1(e1392) embryos and between crn-6(tm890); nuc-1(e1392) and crn-7(ok866) crn-6(tm890); nuc-1(e1392) embryos , crn-6(tm890), and crn-7(ok866) single mutants, and their double or triple mutants was prepared and incubated with a supercoiled plasmid DNA in an acidic condition (pH 5.0) (see crn-6(tm890), or crn-7(ok866) worm lysate displayed a strong endonuclease activity, resulting in complete digestion of the plasmid DNA in 10 minutes at 37°C (nuc-1(e1392) lysate (crn-6(tm890); nuc-1(e1392) animals or crn-7(ok866) crn-6(tm890); nuc-1(e1392) animals, this residual nuclease activity was lost  see . N2, crn) lysate , lane 3,C. elegans. Transcriptional reporter constructs containing the promoter of the DNase II gene fused to GFP with four tandem copies of the nuclear localization signal (NLS) were made and injected into wild-type animals . Weak GFP expression was also detected in the vulva, head and body wall muscle cells , we examined whether they mediate apoptotic DNA degradation in the regions where they are expressed, by scoring TUNEL signals in the head and tail regions of nuc-1(e1392), crn-6(tm890), and crn-6; nuc-1 double mutant embryos. In both nuc-1(e1392) and crn-6(tm890) embryos, the majority of TUNEL signals were seen in the head region and some were seen in the tail region (crn-6(tm890); nuc-1(e1392) embryos, the increased TUNEL signals caused by crn-6(tm890) over those of the nuc-1(e1392) mutant were seen mostly in the head region and some in the tail region. These results indicate that nuc-1 and crn-6 affect apoptotic DNA degradation in both anterior and posterior regions of embryos, despite their restricted expression patterns in the anterior and the posterior regions of the embryo, respectively. This finding is also consistent with the observation that most C. elegans embryonic cell deaths occur in the anterior region of the embryo nuc-1 and crn-6 mutant embryos. The fact that both NUC-1 and CRN-6 affect apoptotic cells outside of their expression areas suggests that these two nucleases may be secreted  L2 larvae treated with Syto 11 showed strong staining of intestine and multiple apoptotic cells in the ventral nerve cord (nuc-1(e1392) late L1 to early L3 larvae had an average 6.5 pycnotic nuclei and 89% of L3-L4 larvae had strong gut staining (crn-6(tm890), crn-7(ok866), or crn-7(ok866) crn-6(tm890) larvae did not contain pycnotic nuclei in the ventral cord and few of them had strong gut staining when stained with Syto 11 (crn-6(tm890) crn-7(ok866), crn-6(tm890); nuc-1(e1392), crn-7(ok866); nuc-1(e1392), or crn-7(ok866) crn-6(tm890); nuc-1(e1392) animals (crn-6(tm890); nuc-1(e1392)/+ or crn-7(ok866) crn-6(tm890); nuc-1(e1392)/+ animals (nuc-1(e1392)/+ heterozygosity could provide a sensitized background for revealing the potential contribution of crn-6 and crn-7 to these two DNA degradation events. Therefore, NUC-1 appears to be the only DNase II nuclease that has a function in degrading DNA in post-embryonic apoptotic cells and bacterial DNA in intestine.In addition to causing accumulation of TUNEL-positive signals in embryos, loss of rve cord , which irve cord . The venstaining . In cont Syto 11 . These r animals . Nor did animals , in whicC. elegans DNase II homologues can functionally substitute for one another. We generated transgenes that drive the expression of NUC-1::GFP, CRN-6::GFP, or CRN-7::GFP under the control of the nuc-1 promoter (Pnuc-1) and then examined whether these fusion proteins can rescue the nuc-1(e1392) defects , no GFP was detected in embryos but weak GFP signals could be observed in the two anterior intestinal cells in larvae (nuc-1(e1392) embryos (nuc-1 promoter used in the transgenes is sufficient to drive nuc-1::gfp expression to rescue the TUNEL defect of the nuc-1(e1392) mutant. In two Pnuc-1crn-6::gfp lines (smIs173 and smIs175), CRN-6::GFP was seen in the head region of embryos  exhibited significant rescue of the TUNEL defect of the nuc-1(e1392) mutant in all three embryonic stages, whereas smIs175 (with 7–8 copies) showed mild rescue in comma and 1.5-fold nuc-1(e1392) embryos and no rescue in 4-fold nuc-1(e1392) embryos  with a higher copy number (31–32 copies) and stronger GFP fusion expression than either Pnuc-1nuc-1::gfp transgenes (smIs170 and smIs172) only achieved partial rescue of the nuc-1 mutant. In three Pnuc-1crn-7::gfp transgenic lines, smIs209–211 , we did not detect obvious GFP signals in embryos or in larvae (nuc-1 TUNEL defect (smEx4085) that contains Pnuc-1crn-7::gfp, although we observed bright CRN-7::GFP signals in the anterior region of the transgenic embryos, we failed to detect rescue of the nuc-1 TUNEL defect ; smIs170 or nuc-1(e1392); smIs172 larvae, whereas an average of 6.7, 6.3, 6.4, and 5.5 pycnotic nuclei posterior to the vulva were observed in different larval stages of the nuc-1(e1392) mutant . Partial rescue of the “pycnotic nuclei” phenotype was observed in nuc-1(e1392) animals carrying a higher copy number of the Pnuc-1crn-6::gfp transgene (smIs173), especially in the late larval stages (nuc-1(e1392) animals at the same stage. Poor or no rescue of the “pycnotic nuclei” phenotype was observed in nuc-1(e1392) animals with a lower copy number of the Pnuc-1crn-6::gfp transgene (smIs175) or with four different Pnuc-1crn-7::gfp transgenes  animals. Two Pnuc-1nuc-1::gfp transgenes, smIs170 and smIs172, can strongly rescue the defect in digesting bacterial DNA , one transgene  showed strong rescue of the gut staining in the nuc-1(e1392) mutant  failed to do so. Therefore, the crn-6 promoter is capable of directing the expression of NUC-1 to mediate degradation of bacterial DNA in intestine but the level of NUC-1 expression appears to be critical for this function.Interestingly, when NUC-1::GFP was expressed under the control of the ) mutant . Howevercrn-6 and nuc-1 do not overlap in embryos (except a few cells at the 4-fold stage embryos) and CRN-6 is expressed specifically in the posterior half of embryos (crn-6 promoter (Pcrn-6nuc-1::gfp) affects DNA degradation in apoptotic cells, which occur mostly in the anterior half of the embryos. In animals carrying two Pcrn-6nuc-1::gfp transgenes (smIs195 and smIs199), no obvious GFP signal was observed in embryos and only weak GFP signals could be seen in the two anterior intestinal cells. In contrast, in animals carrying two Pcrn-6crn-6::gfp transgenes (smIs187 and smIs189) that fully rescued the TUNEL defect of crn-6(tm890) embryos  still resulted in 50% reduction of the TUNEL signals in nuc-1(e1392) comma and 1.5-fold embryos and almost completely rescued the TUNEL defect of nuc-1(e1392) 4-fold embryos , partial rescue of the TUNEL defect of nuc-1(e1392) embryos was detected  larvae, especially in later larval stages  L3–L4 larvae, compared with 5.5 pycnotic nuclei seen in nuc-1(e1392) larvae at the same stage  failed to rescue the pycnotic nuclei phenotype of nuc-1(e1392) larvae.Similar to what was observed in the embryo, l stages . For exame stage . In contmyo-2 gene promoter, which drives gene expression exclusively in the pharyngeal muscle in the head  mutant and partially rescued the defect in degrading intestinal bacterial DNA ::GFP  failed to rescue the pycnotic nuclei phenotype and the defect in degrading intestinal bacterial DNA in the nuc-1(e1392) mutant (nuc-1(e1392) mutant (nuc-1(e1392) mutant  mutant , indicat) mutant . On the ) mutant . AltogetC. elegans NUC-1 is implicated to function in apoptotic cells nuc-1 expressed in posterior intestine cells under the control of the crn-6 promoter can still promote apoptotic DNA degradation in the anterior region of the embryo (smIs195 transgene (Pcrn-6nuc-1::gfp) still rescued the TUNEL defect in ced-2(e1752); nuc-1(e1392) or ced-6(n2095); nuc-1(e1392) embryos, in which cell corpse engulfment is severely compromised, or the TUNEL defect of the ced-6(n2095); ced-2(e1752); nuc-1(e1392) triple mutant, in which both engulfment pathways are blocked and few apoptotic cells are engulfed by phagocytes nuc-1 rescuing activity is not contributed by neighboring engulfing cells. Instead, NUC-1 can act in apoptotic cells to digest 3′OH DNA breaks.In mammals, DNase II is thought to act in engulfing cells, whereas in e embryo . This fiC. elegans, multiple DNase II nucleases exist but their relative contributions to apoptosis and animal development remain unclear. Moreover, how DNase II enzymes act to promote apoptotic DNA degradation remain controversial. In this study, we carried out comprehensive analysis of nuc-1, crn-6, and crn-7, three genes that encode C. elegans DNase II enzymes. Our results indicate that three C. elegans DNase II homologues play differential roles in both apoptotic and non-apoptotic DNA degradation. Strikingly, we discover that NUC-1 is a secreted nuclease that can travel a long distance and be retaken up at distant sites to mediate DNA degradation in apoptotic cells.DNase II enzymes are the major acidic nucleases in cells and have been shown to play important roles in apoptosis and animal development C. elegans and mammals has raised two important questions regarding how they might act to promote DNA degradation C. elegans DNase II gene promoters. We found that the embryonic expression patterns of the three DNase II genes in C. elegans do not overlap, except for a few anterior intestinal cells in 4-fold embryos. In larvae and adults, both nuc-1 and crn-6 are expressed in some intestinal cells and there is partial overlap in nuc-1 and crn-7 expression in the head and body wall muscle cells  or has restricted expression in a few cell types (DNase IIβ) C. elegans.The identification of multiple DNase II homologues in le cells . The larnuc-1, crn-6, or crn-7 causes increased number of TUNEL-positive cells in mutant embryos ; nuc-1(e1392) double mutant is approximately 87% of that in wild type animals or 75–85% of that in individual crn-6, nuc-1, or crn-7 single mutants , suggesting that this defect is not related to apoptosis and that the three DNase II enzymes might have a redundant but important role in germ line development.Although we did not observe an obvious role for CRN-7 in any of the DNA degradation events, loss of  mutants . In contC. elegans DNase II genes, we expressed them under the control of the nuc-1 promoter and examined the ability of each transgene in rescuing the nuc-1(e1392) defects. Despite of an overall sequence similarity between CRN-6 and NUC-1, especially at the catalytic site (nuc-1 promoter (nuc-1(e1392) mutant and completely fails to rescue the defect in degrading bacterial DNA in intestine  or ced-6(lf) mutations clearly does not affect apoptotic DNA degradation mediated by nuc-1in vivo, and how secretion of DNase II might affect its in vivo DNA degradation functions. In our analysis of crn-6 and nuc-1 expression patterns through transcriptional reporters, crn-6 transcription is found to occur specifically in the posterior half of the embryo, whereas nuc-1 is transcribed specifically in the anterior half of the embryo in a largely non-overlapping fashion (crn-6nuc-1::gfp), nuc-1 was expressed in the posterior half of embryos under the control of the crn-6 promoter, yet one of the Pcrn-6nuc-1::gfp transgenes (smIs195) almost completely rescued the TUNEL defect of the nuc-1(lf) 4-fold embryos  DNA degradation defect in apoptotic cells of the posterior ventral cord, which are situated far apart, more than half of the body length, from the head region  into the nuc-1(e1392) mutants that were also defective in both cell corpse engulfment pathways. Surprisingly, smIs195 displayed no discernable difference in rescuing the TUNEL defect of the nuc-1(lf) and ced-6(lf); ced-2(lf); nuc-1(lf) embryos, where cell corpse engulfment is blocked, and completely rescued the TUNEL defect of the ced-6(lf); ced-2(lf); nuc-1(lf) 4-fold embryos  (this study), crn-7(ok866) (C. elegans gene knockout consortium), ced-6(n2095)unc-119(ed3)ced-2(e1752)ced-3(n2433)unc-76(e911)him-5(e1490)nuc-1(e1392)axIs36nuc-1 cDNA from an N2 cDNA library. To construct the nuc-1::gfp vector, nuc-1 coding sequence without a stop codon was PCR amplified from the nuc-1 cDNA clone using primers A1 and A3 and inserted into the pPD95.79 vector (a gift from Andrew Fire). The crn-7 coding sequence without a stop codon was PCR amplified using primers B1 and B2 and a crn-7 RT-PCR product as the template. The crn-6 coding sequence without a stop codon was amplified using primers C1 and C2 from the EST clone yk720e2 (a gift from Dr. Yuji Kohara). They were then cloned into pPD95.79 to generate crn-7::gfp and crn-6::gfp fusion vectors, respectively. All cDNAs were sequenced and confirmed to be identical to sequences reported in the Wormbase, except that the yk720e2 crn-6 cDNA clone contains a base substitution . To obtain the promoter sequences of three DNase II genes, the 5′ 1862 bp upstream region of the crn-6 gene was PCR amplified using primers C3 and C4 and fosmid WRM0621aA08 as a template. The 5′ 3227 bp upstream region of the nuc-1 gene was PCR amplified using primers A4 and A5 and fosmid WRM0611cG07 as a template. And the 5′ 818 bp upstream region of the crn-7 gene was PCR amplified using primers B3 and B4 and fosmid WRM0613aA03 as a template. These promoter fragments were cloned into vector pPD122.56 to generate 4xNLS::GFP transcriptional fusions or cloned into vector pPD95.79 to generate transcriptional GFP fusions. Primers B1 and B5 were used to detect the crn-7(ok866) deletion by PCR and primers C5 and C6 were used to detect the crn-6(tm890) deletion. To construct the nuc-1 cDNA clone without the signal peptide [NUC-1(22–375)], the region encoding NUC-1(22–375) was amplified by PCR using primers A8 and A3 and the nuc-1 cDNA clone as a template. To construct the nuc-1 cDNA clone with a defective signal peptide [NUC-1] and the nuc-1 cDNA clone encoding NUC-1, we performed quick-change mutagenesis using primers A9 and A10 and primers A11 and A12, respectively. The plasmid L3790 was used as the source of the myo-2 promoter. To construct plasmids for ballistic bombardment, various coding regions tagged with GFP were fused with different promoters and the resulting translational fusions were then cloned into the PBSKrt2-unc-119 bombardment vector (a gift from Dr. Min Han).Sequences of primers described in this section are shown in crn-64xNLS::GFP at 60 µg/ml and the pBluescript II SK vector at 40 µg/ml (as carrier DNA). To study the expression patterns of NUC-1 and CRN-7, N2 animals were injected with Pnuc-14xNLS-GFP at 40 µg/ml or Pcrn-74xNLS::GFP at 60 µg/ml along with pRF4 [containing the dominant marker rol-6(su1006)] at 40 µg/ml as a transgenic marker. nuc-1(e1392) animals were injected with 60 µg/ml of Pnuc-1crn-7::gfp and 40 µg/ml of Pmyo-2gfp (as a transgenic marker) to generate smEx4085. To express various NUC-1 mutants under the control of the myo-2 promoter, nuc-1(e1392) animals were injected with Pmyo-2NUC-1 (50 µg/ml), Pmyo-2NUC-1(22-375)::GFP (50 µg/ml), Pmyo-2 NUC-1 (60 µg/ml), or Pmyo-2NUC-1 (50 µg/ml), using Pmyo-2gfp (50 µg/ml) as a co-injection marker. To detect the GFP expression patterns of Pmyo-2NUC-1::GFP and Pmyo-2NUC-1(22-375)::GFP, N2 animals were injected with Pmyo-2NUC-1::GFP (50 µg/ml) or Pmyo-2NUC-1(22–375)::GFP (50 µg/ml) along with pRF4 at 50 µg/ml. Integrated transgenic lines, smIs170, smIs172, smIs173, smIs175, smIs209–211, smIs187, smIs189, smIs195 and smIs199  were added into the microfuge tube to anesthetize the worms, which were immediately observed by fluorescence microscopy with FITC filter. The pycnotic nuclei in the posterior ventral cord of the animals, from vulva to anus, were scored in late L1 to early L3 larvae unless otherwise noted. For the intestine staining, L3 to L4 larvae were scored.The vital DNA-binding dye Syto 11  was used to stain the condensed chromosomal DNA in the ventral cord apoptotic cells and the undigested bacteria DNA in the gut lumen C. elegans lysates were performed essentially as described previously C. elegans strains were prepared by sonication in lysis buffer  containing protease inhibitors . After sonication, Triton X-100 was added to 1% and the lysates were incubated on ice for 30 min. Protein concentrations of the supernatants derived from the crude lysates were determined at OD595 using a Dye Reagent Concentrate (Bio-Rad). One µg of each worm extract was incubated with 750 ng of the pSL1190 plasmid DNA at 37°C for 10 min (in 50 mM acetic acid pH 5.0) in a 30 µl total volume. Samples were extracted using phenol: chloroform: isoamyl alcohol (25:24:1)  to remove proteins. 20 µl of each sample were then analyzed on a 1% agarose gel and stained with EtBr. CstF-64 was used as a loading control, based on western blot analysis of 30 µg of lysates using anti-CstF-64 antibody .The DNase activity assays from A Zeiss Axioplan 2 microscope with a 100x optic was used to score the cell corpses in the embryos. The same 100x oil immersion optic was used with a FITC filter to score the TUNEL staining in embryos and the Syto 11 staining in larvae and with a Rhodamine filter to score the anti-GFP antibody staining in embryos. The microscope was equipped with a SenSiCam CCD camera  and Slidebook 4.0 software .crn-6 gene. Primers A6 and A7 were used to determine the copy number of the nuc-1 gene. Primers B6 and B7 were used to determine the copy number of the crn-7 gene. The lmn-1 gene amplified by primers D1 and D2 was used as an internal control.Quantitative PCR was performed using a Rotor-gene 3000 machine  and SYBR Green JumpStart . Sequences of primers are described in Figure S1C. elegans DNase II genes and schematic representation of DNase II mutants. A) Sequence alignment of two human DNase II nucleases and three C. elegans DNase II homologues. Residues that are identical are indicated by red and residues that are similar are indicated by blue. Arrowheads indicate potential cleavage sites of signal peptides as predicted by the SignalP 3.0 program , except that the cleavage site in human DNase IIα was determined experimentally. The box indicates the catalytic site of DNase II crn-6 and crn-7 genes and the e1392 mutation in nuc-1. Gray boxes represent exons and waved lines indicate introns. The actual size of each deletion  is indicated below by the black boxes.Alignment of human and (1.80 MB TIF)Click here for additional data file.Figure S2nuc-1 and wild-type animals. DIC and Syto 11 fluorescent images of a L2 larva are shown. Arrowheads indicate pycnotic nuclei in the posterior ventral cord of the nuc-1(e1392lf) animal. Black arrowhead indicates the undigested bacterial DNA in the gut that is strongly stained by Syto 11. Scale bars indicate 5 µm.Syto 11 staining of (2.39 MB TIF)Click here for additional data file.Figure S3nuc-1crn-6::gfp animals. DIC and GFP images of smIs173 transgenic animals at different developmental stages: (A) an early embryo, (B) a comma stage embryo, (C) a 2-fold stage embryo, (D) a 4-fold stage embryo,  larvae, and (G) adult. The head region in A–D and F are indicated by brackets. Many GFP signals were observed in the head region. In E and F, arrowheads indicate the two most anterior intestinal cells and arrows indicate the most posterior intestinal cells. Arrows in H indicates the gut lumen. The square in G indicates the vulva region. Scale bars indicate 5 µm .The GFP expression patterns of P(3.68 MB TIF)Click here for additional data file.Figure S4crn-6crn-6::gfp animals. A) TUNEL analysis of crn-6(tm890) embryos carrying smIs187 and smIs189 transgenes (Pcrn-6crn-6::gfp). At least 30 embryos from each embryonic stage were scored. Error bars indicate SEM. (B–F) The GFP expression patterns of smIs187 animals. DIC and GFP images of smIs187 animals at various developmental stages are shown: B) an early embryo, C) a comma embryo, D) a 2-fold embryo, E) a 4-fold embryo, and F) a larva. Arrowheads in E and F indicate the most anterior intestinal cells. Scale bars indicate 5 µm.TUNEL assay and the GFP expression patterns of P(8.07 MB TIF)Click here for additional data file.Table S1Copy number and GFP expression in various integrated lines generated by ballistic bombardment(0.04 MB DOC)Click here for additional data file.Table S2The crn-7 crn-6; nuc-1 triple mutant has a smaller brood size than N2 animals or any of the single mutants.(0.03 MB DOC)Click here for additional data file.Table S3Primers used in this study(0.05 MB DOC)Click here for additional data file.


ErbB2, a member of the epidermal growth factor receptor (EGFR) family, is overexpressed in 20% to 30% of human breast cancer cases and forms oncogenic signalling complexes when dimerised to ErbB3 or other EGFR family members.We crossed mouse mammary tumour virus (MMTV)-myr-Akt1 transgenic mice (which express constitutively active Akt1 in the mammary gland) with MMTV-c-ErbB2 transgenic mice to evaluate the role of Akt1 activation in ErbB2-induced mammary carcinoma using immunoblot analysis, magnetic resonance spectroscopy and histological analyses.Bitransgenic MMTV-c-ErbB2, MMTV-myr-Akt1 mice develop mammary tumours twice as fast as MMTV-c-ErbB2 mice. The bitransgenic tumours were less organised, had more mitotic figures and fewer apoptotic cells. However, many bitransgenic tumours displayed areas of extensive necrosis compared with tumours from MMTV-c-ErbB2 mice. The two tumour types demonstrate dramatically different expression and activation of EGFR family members, as well as different metabolic profiles. c-ErbB2 tumours demonstrate overexpression of EGFR, ErbB2, ErbB3 and ErbB4, and activation/phosphorylation of both ErbB2 and ErbB3, underscoring the importance of the entire EGFR family in ErbB2-induced tumourigenesis. Tumours from bitransgenic mice overexpress the myr-Akt1 and ErbB2 transgenes, but there was dramatically less overexpression and phosphorylation of ErbB3, diminished phosphorylation of ErbB2, decreased level of EGFR protein and undetectable ErbB4 protein. There was also an observable attenuation in a subset of tyrosine-phosphorylated secondary signalling molecules in the bitransgenic tumours compared with c-ErbB2 tumours, but Erk was activated/phosphorylated in both tumour types. Finally, the bitransgenic tumours were metabolically more active as indicated by increased glucose transporter 1 (GLUT1) expression, elevated lactate production and decreased intracellular glucose (suggesting increased glycolysis).Expression of activated Akt1 in MMTV-c-ErbB2 mice accelerates tumourigenesis with a reduced requirement for signalling through the EGFR family, as well as a reduced requirement for a subset of downstream signaling molecules with a metabolic shift in the tumours from bitransgenic mice. The reduction in signalling downstream of ErbB2 when Akt is activated suggest a possible mechanism by which tumour cells can become resistant to ErbB2-targeted therapies, necessitating therapies that target oncogenic signalling events downstream of ErbB2. The ErbB2/Neu/HER2 oncogene is amplified and overexpressed in 20% to 30% of human breast cancer cases, and expression of ErbB2 is associated with aggressive metastatic tumour behaviour, decreased time to clinical relapse and poor prognosis ,2. The iet al. , 1 mM dithiothreitol, 5 mM sodium orthovanadate, 100 μg/ml phenylmethanesulphonylfluoride and a complete protease inhibitor cocktail ), followed by Dounce homogenisation. Lysates were clarified by centrifugation and protein concentrations determined using the Bradford assay . Equal amounts of total protein per lane (2 to 50 μg) were resolved on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membrane  and immunoblotted with the desired antibody.Anti-HA antibody was obtained from Roche Applied Sciences . Anti-phospho-ErbB2 (Tyr877), anti-phospho-ErbB3 (Tyr1289), anti-phospho-retinoblastoma (Rb) (Ser780), anti-phospho-Akt (Ser473), anti-phospho-Src (Tyr416), anti-phospho-Gab2 (Tyr452), anti-phospho-Shc (Tyr313), anti-phospho-Shc (Tyr239/240), anti-Akt, anti-p15, anti-p27 and anti-cyclin D1 antibodies were obtained from Cell Signaling Technologies . The anti-ErbB2, anti-ErbB3, anti-ErbB4, anti-EGFR, anti-Src, anti-Shc, anti-β-actin and anti-ERK1 antibodies were obtained from Santa Cruz Biotechnology . The anti-phospho-ErbB2 (Tyr1248) was obtained from Abcam . The anti-Rb antibody was obtained from BD Pharmingen . The anti-phospho-ERK antibody was obtained from Promega . The anti-Gab2 polyclonal antibody was generously supplied by Dr. Haihua Gu . The glucose transporter 1 (GLUT1) polyclonal antibody was generated by Global Peptide  by immunising rabbits with a peptide corresponding to the C-terminus of the human/mouse GLUT1 sequence (KTPEELFHPLGADSQV) and affinity purifying the resulting IgG.1H-magnetic resonance spectroscopy (MRS) with the Bruker narrow-bore 500 MHz DRX system and an inverse TXI-5-mm probe . The following 1H-nuclear magnetic resonance (NMR) parameters with water suppression ('zgpr') were used: 500.12 MHz operating 1H frequency; 40 accumulations; 0 dB power level; 55 dB irradiation power level; 7.5 μs pulse width; 12 ppm spectral width; and 12.8 second repetition time (fully relaxed). An external standard substance, trimethylsilyl propionic-2,2,3,3,-d4 acid ([TMSP] 20 and 50 mM in heavy water) in a thin sealed glass capillary was placed into the NMR tubes during all experiments for metabolite quantification and as a 1H chemical shift reference (at 0 ppm). After performing Fourier transformation, phase and base line corrections, each 1H peak of corresponding metabolites was integrated using 1D WINNMR program . The NMR peak assignment was confirmed by two-dimensional NMR spectra . Th. Th36]. Most tumour cells rely on increased glycolysis, even in the presence of available oxygen (the Warburg effect). It has been shown that p53, HIF-1 (hypoxia inducible factor 1), c-Myc as well as Akt can all upregulate glycolytic enzymes  to trigger increased tumour cell glucose consumption . The incet al. demonstrated that loss of the PTEN tumour suppressor (which induces Akt activation) predicts resistance to trastuzumab treatment [Human breast carcinomas overexpress ErbB2 in 20% to 30% of cases and patients with this type of tumour bear a poor prognosis and are currently treated with trastuzumab, a humanised monoclonal antibody. Trastuzumab treatment significantly boosts the prognosis for patients with breast tumours that overexpress ErbB2, but the response rate for these patients is about 50% to 60% . Nagata reatment . We predExpression of activated Akt1 in the mammary gland of MMTV-c-ErbB2 mice accelerates tumourigenesis and attenuates signalling events sometimes thought to be critical to tumourigenesis. The bitransgenic tumours also have an accelerated glucose metabolism. Our studies suggest that tumours that overexpress ErbB2 which activate Akt by means of mutating PI3K, PTEN or Akt (rather than via ErbB2/ErbB3 activation of PI3K) may be resistant to ErbB2-targeted therapies. Therefore, therapies which molecularly target signalling events downstream of ErbB2, such as those mediated by Akt, may prove to be valuable.ANOVA = analysis of variance; Akt1DD = constitutively active Akt1 mutant bearing two phospho-mimetic mutations; EGFR = epidermal growth factor receptor; ERK = extracellular-signal regulated kinase; FVB = inbred mouse strain which was exclusively used in this study; Gab2 = Grb2 associated binding protein 2; GLUT1 = glucose transporter 1; L2 = lactation day 2; HIF1 = Hypoxia inducible factor 1; MMTV = mouse mammary tumour virus; MRS = magnetic resonance spectroscopy; Myr-Akt1 = constitutively active Akt1 mutant bearing the myristolation sequence from Src; NMR = nuclear magnetic resonance; PI3K = phosphatidyl inositol-3 kinase; PTEN = phosphatase and tensin homologue deleted on chromosome 10; PVDF = polyvinylidene difluoride; Rb = retinoblastoma; SD = standard deviation; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; Shc = Src homology 2 domain-containing transforming protein C1; TMSP = trimethylsilyl propionic-2,2,3,3,-d4 acid.The authors declare that they have no competing interests.ERBB2 mutation analysis and assisted with preliminary studies. AL maintained the animal colony including breeding, genotyping, tumour palpation and tissue harvest. NJS performed MRS analysis and interpreted metabolic data. SMA conceived the study and directed the research. NJS and SMA critically reviewed the manuscript, which was approved by all authors.CDY performed the immunoblot analyses, interpreted data, assembled the figures and drafted the manuscript. EN quantitated apoptosis, performed


To evaluate the diagnostic application of urinary transforming growth factor-beta1 (TGF-beta1) and serum alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC), TGF-beta1 and AFP were determined in 94 patients with cirrhotic HCC and in 94 sex- and age-matched patients with cirrhosis alone. TGF-beta1 and AFP levels in HCC were higher than in cirrhosis alone (P = 0.0001). There is an inverse correlation between TGF-beta1 and log AFP . Multivariate analysis indicated that TGF-beta1 and AFP were closely associated, in a dose-related fashion, with the development of HCC. Receiver-operating characteristic (ROC) curves were used to determine the optimal cut-off values of TGF-beta1 (50 microg g(-1) creatinine) and AFP (100 ng ml(-1)). Both TGF-beta1 and AFP showed a high specificity (99%) and positive likelihood ratio. The sensitivity was 53.1% for TGF-beta1 and 55.3% for AFP. The determination of both markers in parallel significantly increased the diagnostic accuracy (90.1%) and sensitivity (84%), with a high specificity (98%) and positive likelihood ratio (40.0). In conclusion, TGF-beta1 and AFP are independent tumour markers of HCC and may be used as complementary tumour markers to discriminate HCC from cirrhosis.


High mobility group box-1 (HMGB-1) is a DNA-binding protein secreted by activated monocytes and has been identified as a key late mediator of endotoxic shock. We investigated the regulation of HMGB-1 in human peripheral blood mononuclear cells (PBMCs) following stimulation with the staphylococcal superantigen, toxic shock syndrome toxin-1 (TSST-1), and found that TSST-1, like LPS, induced the secretion of HMGB-1 from human PBMC. However, unlike monocyte-driven sepsis caused by endotoxin, translocation and secretion of HMGB-1 mediated by TSST-1 was dependent on the presence of both activated T cells and monocytes. Furthermore, we show that nuclear HMGB-1 is released from TSST-1 stimulated T cells. This finding presents a basis for investigating the potential of targeting HMGB-1 for the treatment of toxic shock syndrome, and provides further insight on the role of HMGB-1 in the cross-talk between activated monocytes and T cells. Furthermore, neutralizing antibodies directed atHMGB-1 rescued mice from lethal endotoxemia even when administered 24 hoursafter sepsis initiation [High mobility group box-1 (HMGB-1) protein is a30 kDa nonhistone nuclear DNA binding protein that is shown to have anextracellular role in inflammation, cell differentiation, adherence, andmotility . HMGB-1 itiation , 9. Foritiation .S. aureus, and this sAgwas subsequently identified as the majorcausative agent of toxic shock syndrome [β-specific regions of the αβ T cell receptor (TCR) to class II major histocompatibility complex (MHC II) molecules on antigen presenting cells (APCs) [α [Toxic shock syndrome toxin-1 (TSST-1) is asuperantigen (sAg) secreted by some strains of syndrome . In cons (APCs) . In addis (APCs) , which aPCs) [α , 7, 14, S. aureus strain RN4220 previously transformed to carry the tst gene,using both preparative isoelectric focusing and chromatofocusing [Limulus amoebocyte lysate gelation.Recombinant TSST-1 was purified from culture supernatants of ocusing . Toxin p6 cells/mL in complete culture mediumconsisting of RPMI 1640 ,10% heat-inactivated fetal bovine serum , 2 mM L-glutamine (StemCell), 25 mM Hepes buffer (StemCell), and 2 ug/mLof polymyxin B sulphate . For analysisof secreted HMGB-1 in cell culture supernatants, PBMCs were plated in 24-wellflat-bottom plates in Opti-MEM I reduced serum medium (Gibco). THP-1 cells(human monocytic cell line) were obtained from ATCC and kept in recommendedgrowth media until use, at which time the cells were plated at a concentrationof 1.5 × 106 cells in Opti-Mem I medium. T cell depletion of humanPBMC was performed using a column-free method of magnetic bead separation(EasyCep from StemCell Technologies) by positive selection of T cells using ananti-CD3 conjugated antibody according to manufacturer's directions. Thisprocedure left the monocytes negatively selected for and untouched for the purposeof further experimental investigations.Fresh human PBMCs from healthy donors were obtained by Ficoll-Paque PLUS  density centrifugation, and cultured in 96-wellU-bottom plates at 1.5 × 102 prior to stimulation with 1 nM TSST-1 or 500 ng/mL LPS (Sigma), where noted. Culture supernatants were collected at 24 hourspost-TSST-1 treatment, microcentrifuged at 800xg for 5 minutes, and frozen at –70°C until protein analysis.Purified PBMCs were plated in either 24-well or 96-well culture plates overnight at37°C in 5% CO2) directly on coverslips to allow attachment ofadherent cells overnight before stimulation with 1 nM of TSST-1. Cells werethen fixed and surface-stained as above (but without the permeabilizationstep). Fluorescent-labeled PBMCs were subsequently stained with Hoechst 3342nuclear dye (molecular probes), and mounted on slides using prolong antifadereagent (molecular probes). Cells were visualized with an AxioPlan IIfluorescence microscope equipped with a CCD camera using Northern Eclipsesoftware (Epix) for acquisition of images. Images were taken with the 63x oilimmersion objective lens, and Adobe Photoshop 6.0 software was used for imagelayout.PBMCs were first surface-stained for expression of CD3 with mouseantihuman CD3 IgG  followed by antimouse IgG antibodyconjugated to Alexa594 (Molecular Probes); cells were subsequently fixed usingCytofix buffer (Pharmingen). For the intracellular detection of HMGB-1, cellswere permeabilized with Cytoperm (Pharmingen) and incubated for 30 minutes(4°C) with rabbit polyclonal anti-HMGB-1 antibodies  diluted in blocking buffer followed by a secondary antibody with Alexa488-conjugatedgoat antirabbit IgG. The anti-HMGB-1 antibodies were raised against the peptidesequence corresponding to amino acids 166–181 that were previously shown to bespecific for HMGB-1 and not HMGB-2 [Surface-expressed HMGB-1 was analyzed by flow cytometry  using the anti-HMGB-1 antibody andsecondary Alexa488-conjugated antibody described above in conjunction withphycoerythrin (PE)-conjugated anti-CD3 and anti-CD14 antibodies (BD BioSciencesPharmingen), with a minimum of 10 000 events collected for each sample.PBMC or THP-1 culture supernatants were concentrated 10-fold from original volume usingAmicon Ultra centrifugal filters with a molecular weight cut-off of 10 kDa(Millipore). Some culture supernatantshad been further prepared using the SDS-PAGE Clean-Up Kit (Amersham) accordingto the manufacturer's directions prior to running on a 12% polyacrylamide gel.Western blotting was performed by semidry transfer of proteins  onto an Immobilon-P PVDF membrane (Millipore) which had beenblocked for 1 hour at room temperature with 1% BSA, 0.5% Tween in Tris bufferedsaline (TBS) prior to overnight incubation (at 4°C) with rabbit polyclonalanti-HMGB-1 antibody. The membrane was subsequently incubated with antirabbitIgG horse-radish-peroxidase (HRP)-conjugated secondary antibody for 1 hour atroom temperature on a shaker. Detection of HMGB-1 was performed using super signalsubstrate (Pierce) and developed as well as analyzed using the Alpha Innotech3400 Gel Documentation system .t-test.Differences were considered significant if P < .05. Statistical analysis was performed using Prism3.0 software package (GraphPad). The proportion of PBMCs expressing HMGB-1following treatment with TSST-1 or RPMI in different donors was compared by paired Student's P < .05, paired Student's t-test from4 different donors). Flow cytometric analysis, to our knowledge, has not been utilized previously for quantifying the surface expression ofHMGB-1, and we found it to be a useful tool to evaluate the change inextracellular HMGB-1 expression.Somedifferentiated as well as adherent cells express HMGB-1 on the cell surface, and this cell membrane-associated HMGB-1 has been referred to asamphoterin to distinguish it from intracellular HMGB-1 . Extracγ or TNFα [To investigate the potential change inintracellular HMGB-1 expression upon TSST-1 exposure, human PBMCs were treatedwith 1 nM of TSST-1 for 10 hours, and then subsequently evaluated for thetranslocation of intracellular HMGB-1 by fluorescence microscopy . As repor TNFα , 7 as sTo verify thatHMGB-1 was actively secreted from TSST-1 treated PBMCs, we performed a Westernblot analysis of cell culture supernatants collected 24 hours post-TSST-1treatment .α and IL-1β from human PBMC stimulated with highly purified TSST-1 [Undifferentiated and nonadherent monocytes in suspension do not express cell-surface-associatedHMGB-1. However, during the first steps in differentiation, monocytes activelytransport this molecule to their cell surface, and HMGB-1 is subsequentlysecreted into the extracellular milieu upon further stimulation . To furtd TSST-1 .Various cell types have recently been identified as contributing to the extracellularpool of HMGB-1, including human umbilical vein endothelial cells , plateleβ2-specific T cells and MHC class II moleculesbearing antigen presenting cells, will provide a useful means to study the role of HMGB-1 in T cell-monocyte interactions, Th1polarization, and the ensuing immune response leading to tissue injury. Finally, this study also provides a rationale to investigate thepotential to target HMGB-1 for therapy for inflammatory disorders induced byTSST-1, such as toxic shock syndrome, as it is currently being investigated forgram negative sepsis [The best studied ligand for extracellular HMGB-1 is RAGE (receptor of advancedglycation end products) which is upregulated on activated macrophages as wellas endothelial cells . Previoue sepsis . It shoue sepsis , and it