Osteopontin (OPN) has been shown to play many roles in the progression of cancer. We have recently demonstrated the activation of Akt by OPN. Integrin-linked kinase and PI3-kinase are integral proteins in OPN/AKT pathway in PC3 cells. To investigate the role of the extracellular receptors in OPN signaling, we have examined the spatio-temporal regulation of CD44 and integrin αvβ3 receptor in OPN-induced Akt activation in PC3 cells.Vβ3 integrin or the CD44 cell surface receptor. Members of the Mitogen Activated Protein Kinase (MAPK) family have been shown to be up-regulated in a variety of human cancers and have been implicated in the metastatic behavior. Our studies have demonstrated an increase in the phosphorylation of c-Raf at Ser259 and Ser338 in PC3 cells over-expressing OPN. This increase matches up with the Erk1/2 phosphorylation at Thr202/204 and activation. However, the inhibition of Akt activity augments the phosphorylation state of ERK1/2 to two to three fold with a concomitant reduction in the phosphorylation state of c-Raf at Ser259.Here, our studies demonstrate that OPN can activate Akt either through the αRegulation c-Raf phosphorylation at Ser259 has a role in the anti-apoptotic pathways mediated by Akt or Raf/MEK/ERK proteins. OPN may have dual effects in the activation of Erk1/2. We propose this based on the observations that while OPN activates c-Raf and Erk1/2; it also acts to inhibit c-Raf and Erk1/2 activation through Akt pathway. Our observations suggest that the activation of c-Raf-ERK cascade may promote cell cycle arrest in prostate cancer cells and OPN signaling has a role in the anti-apoptotic mechanism. Osteopontin (OPN) is a multifunctional glycoprotein expressed by a number of cell types. Osteopontin expression has been linked to tumorigenesis and metastasis in a wide range of cancer types including prostate, breast, colon, melanoma, and lung . Tumor bOsteopontin mediates biological function through signal transduction by binding to the cell surface receptors such as integrin αvβ3 and CD44 . It is aThe CD44 family of receptors regulates in a manner similar to that of integrins in cellular responses including adhesion, migration, and the stimulation of both cancerous and non-cancerous cells ,14. Our The Erk pathway is one of the best studied MAPK pathways in mammals and has been shown to be deregulated in approximately one-third of all human cancers . Erk1/2 Raf is targeted to the plasma membrane upon activation by a small GTPase. Phosphorylation of c-Raf at serine 259 is an inhibitory event occurring through Akt . PreviouWe measured the phosphorylation state of the three most widely known members of the mitogen-activated kinase (MAPK) family proteins including Erk1/2, JNK, or p38 MAPK in PC3 cells over expressing OPN (PC3/OPN). Stable PC3/OPN cells were generated as described previously . PC3/OPNImmunoblotting analyses demonstrated a small increase in the phosphorylation of JNK at Threonine 183 and Tyrosine 185 in PC3/OPN cells Figure , lane 2.Raf and MEK have been shown to be the upstream regulators of Erk1/2 . In ordeRecent observations have demonstrated an increase in the activation of Akt in PC3/OPN cells ,25. LittIntegrin αvβ3 and CD44 are receptors of osteopontin and CD44 is frequently over expressed in cancer cells . To asseCD44 is another receptor for OPN  and prevThe ability of OPN to induce phosphorylation and activation of Erk1/2 represents a novel and important signaling mechanism in prostate cancer progression. Here we have identified that the increased expression of OPN leads to the activation of the Erk1/2  PC3 cell lines (over-expressing either full-length or mutated (RGDΔRGA) OPN) in figure n Figure . OPN binAndrogen-independent advanced prostate cancer cell lines such as DU145 and PC3 typically express low levels of activated Raf, MEK, and ERK . In contMonoclonal rabbit anti-phospho-p44/42MAPK (Erk1/2) (Thr202/Tyr204), anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-phospho-c-Raf (Ser338), anti-p44/42MAPK (Erk1/2), anti-B-Raf, polyclonal rabbit anti-phospho-p38MAPK (Thr180/Tyr182), anti-phospho-c-Raf (Ser259), anti-phospho-c-Raf (Ser289/296/301), anti-phospho-A-Raf (Ser299), anti-phospho-B-Raf (Ser445), anti-p38MAPK, anti-SAPK/JNK, anti-A-Raf, and anti-c-Raf were purchased from Cell Signaling Technology . GAPDH and CD44 antibodies were purchased from Santa Cruz Biotechnology Inc . OPN antibody was purchased from Rockland Immunochemicals . Roswell Park Memorial Institute-1640 (RPMI-1640) media, fetal bovine serum (FBS), penicillin-streptomycin , 0.25% Trypsin-EDTA, and phosphate buffered saline (PBS) pH 7.4 were purchased from Invitrogen . Akt (5 μM) inhibitor, rhodamine phalloidin, and other chemicals were purchased from Sigma-Aldrich . Protein assay reagent kit, reagents for polyacrylamide gel electrophoresis (PAGE), and molecular weight standards were purchased from Bio-Rad . Polyvinyldifluoride (PVDF) membrane for immunoblotting analysis was obtained from Millipore Corp. .Stable prostate cancer cell (PC3) lines that either over express unmutated OPN (PC3/OPN) or a mutant OPN in the integrin-binding site (PC3/RGDΔRGA) was generated as described previously . PC3 celCells were washed two times with cold PBS and lysed in ice-cold RIPA lysis buffer . After incubating on ice for 10 min, lysates were centrifuged for 5 min at 6,000 rpm at 4°C. The supernatants were saved and protein concentrations were measured using the Bio-Rad protein assay reagent kit.PC3 cell lines  were cultured in a 6-well culture dish and then treated with one of the following inhibitor in the presence of RPMI-1640 media containing 10% FBS at 37°C: Akt inhibitor for 16 h (1:500 dilution of 5 μM stock), αV integrin inhibitor , siRNA to CD44 (200 nM for 48 h). CD44 siRNA (product number sc-29342) and scrambled siRNA (product number sc-37007) nucleotides were purchased from Santa Cruz Biotechnology Inc . siRNA transfection reagent, RNAiFect, was purchased from Qiagen . Protein lysates were subjected to 12% SDS-PAGE and Western blot analysis as described below.Cell lysates were denatured by boiling for 5 minutes in Novagen 1× SDS sample buffer . Proteins were resolved by SDS-polyacrylamide gel electrophoresis on 8% or 12% gels and then transferred to PVDF membranes. The membranes were initially blocked with PBS containing 0.05% TWEEN 20 (PBS-T) and 5% BSA for 1 h at room temperature (RT) and were then probed overnight at 4°C using a dilution of 1:1000 with the following primary antibodies in PBS-T and 5% BSA: anti-phospho-p44/42MAPK (Erk1/2) (Thr202/Tyr204), anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-phospho-c-Raf (Ser338), anti-phospho-p38MAPK (Thr180/Tyr182), anti-phospho-c-Raf (Ser259), anti-phospho-c-Raf (Ser289/296/301), anti-phospho-A-Raf (Ser299), and anti-phospho-b-Raf (Ser445). After three washes with PBS-T for 5 minutes each, the membranes were incubated with a 1:1000 dilution of species-specific horseradish peroxidase (HRP)- linked secondary antibody  in PBS-T and 5% blotting grade blocker non-fat dry milk  for 2 h at RT. Blots were washed three times with PBS-T for 15 min. each. Protein bands were visualized by chemiluminescence using a SuperSignal West Pico Chemiluminescent Substrate Kit . PVDF membranes were stored in PBS-T at 4°C until being stripped and re-probed with the corresponding control antibodies to determine the loading in each lane as described below.The PVDF membranes were incubated in stripping buffer (2% sodium dodecyl sulfate (SDS), 62.5 mM Tris HCl pH 7.2, and 100 mM β-mercaptoethanol) at 55°C for 15 min. After three washes with PBS-T for 15 minutes each, the membranes were blocked with PBS-T and 5% blotting grade blocker non-fat dry milk  for 1 h at room temperature (RT) and were then probed overnight at 4°C using a dilution of 1:1000 of the primary antibody of interest (e.g. anti-GAPDH at 1:1000 dilution) in PBS-T and 5% blotting grade blocker non-fat dry milk. The membranes were washed three times with PBS-T for 5 min each and were then incubated with a 1:1000 dilution of species-specific horse-radish peroxidase (HRP-) linked secondary antibody in PBS-T and 5% blotting grade blocker non-fat dry milk  for 3 h at RT. Membranes were washed and proteins bands were visualized as described above.PC3 and PC3/OPN cells were cultured onto cover slips in a 12-well dish for 14-16 h at 37°C. Cells were washed three times with room temperature PBS (RT-PBS) and fixed in 4% formaldehyde-PBS for 10 min. After washing three times with RT-PBS, cells were permeabilized with 0.5% Triton X-PBS for 10 min. Cells were washed three times with RT-PBS, followed by incubation in 5% boiled goat serum for 1 h at RT. After washing three times with RT-PBS, cells were incubated with a 1:100 dilution of anti-phospho-p-44/42(ERK1/2) (Thr202/Tyr204) in 5% boiled goat serum overnight at 4°C. Cells were washed three times with RT-PBS. Subsequently, cells were incubated for 3 h at RT in the dark with the following: 1:1000 dilution of FITC-conjugated species specific secondary antibody and 1:500 dilution of rhodamine phalloidin for actin staining. Cells were washed three times with RT-PBS for 15 minutes each and the cover slips were transferred cell side down onto glass slides containing perma fluor mounting medium  and sealed with clear nail polish around the edge of the cover slips. The immunostained cells were viewed and photomicrographed on a Bio-Rad 6000  confocal microscope. Images were stored in TIF image format and processed by the Adobe Photoshop software program .OPN: Osteopontin; RGD: arginine-glycine-aspartic acid sequence. It is known as integrin binding motif; αvβ3: vitronectin receptor; sCD44: soluble or standard CD44; PC3/OPN: PC3 cells over expressing osteopontin; PC3/RGA; PC3 cells expressing mutated OPN in RGD Δ RGA motif where aspartic acid (D) Δ Alanine (A); ERK: extracellular signal regulated kinase; MAPK: mitogen activated protein kinase; FBS: fetal bovine serum; GAPDH: glyceraldehydes-3-phosphate dehydrogenase; FITC: fluorescein isothiocyanateThe authors declare that they have no competing interests.BWR and MAC participated in the design of the study and wrote the manuscript. BWR and LB carried out the experiments. All authors read and approved the final manuscript.


Targeting Dendritic Cells (DCs) with anti-DC receptor antibody-antigen fusion proteins is a novel approach in vaccine development for inducing potent humoral and cellular immune responses.We engineered an anti-human DCIR recombinant antibody cross-reacting with the cynomolgus macaque receptor fused via the heavy chain C-terminus to HIV-1 Gagp24 protein (anti-DCIR.Gagp24). HIV patient PBMC cultures were incubated with anti-DCIR and control hIgG4.Gagp24 fusion proteins. After 10 days, the total T cells were challenged with HIV Gagp24 peptide pools, and then antigen-specific cytokine production was detected using intracellular staining. Macaques were also immunized i.d. 3 times with anti-DCIR.Gagp24 or control hIgG4.Gagp24 with or without polyI:C. Gagp24-specific IgG titers from serum were measured by ELISA and the magnitude of the antigen-specific IFNγ responses was assessed by ELISPOT.In vitro, low doses of anti-DCIR Gagp24 prototype vaccine, but not the control hIgG4.Gagp24, expand of Gagp24-specific T cells. These in vitro-expanded antigen-specific T cells were multifunctional, simultaneously producing multiple cytokines . In vivo, in non-adjuvanted naïve animals, serum anti-Gagp24 antibodies were detectable 2 weeks after priming and titers were substantially increased after the 1st and the 2nd boost with anti-DCIR.Gagp24 and were durable, while in the control hIgG4.Gagp24 group the responses were minimal. Poly I:C increased the magnitude of the responses in the anti-DCIR.Gagp24 and hIgG4.Gagp24 groups to a similar level in both groups. T cell responses induced by anti-DCIR Gagp24 could be enhanced after priming with a recombinant modified vaccinia virus Ankara (MVA) encoding HIV Gag/Pol/Nef. Boosting with anti-DCIR.Gagp24 plus poly I:C generated high titers of anti-Gagp24 antibody titers and strongly enhanced IFNγ-producing T cells following priming with MVA HIV Gag/Pol/Nef.Our results demonstrate that heterologous prime-boost immunization with vectors and DC-targeting protein-based vaccines is a promising vaccination approach to optimize humoral and cellular immunity for therapeutic and preventive applications against AIDS.


Transmitted viral factors have been shown to affect disease progression but whether infection with viruses carrying beneficial HLA-mediated escape polymorphisms affects disease progression in HLA-mismatched participants remains controversial.Gag was sequenced from 56 participants with acute HIV infection from the CAPRISA 002 cohort. For the newly identified mutation, replication fitness of viruses in PBMCs was compared in a subtype C infectious molecular clone. Disease progression was compared in participants infected with viruses carrying polymorphisms associated with beneficial HLA- B*57/58:01 and B7 supertype (B*39:10/81:01) allelic selective pressure in HLA mismatched participants.In HLA-B*57/58:01-negative participants, 51% (25/49) were infected with viruses carrying at least one of the B*57/58:01 fitness cost-associated escape mutations in ISW9 (A146P:19/49), KF11 (A163G: 10/49) or TW10 (T242N/S: 11/49) epitope. In HLA-B7 negative participants, only 9.4% (3/32) had viruses carrying mutations in the HLA-B7 immunodominant TL9 epitope. Furthermore, we identified in viruses from HLA-B7 participants and included in this analysis a novel mutation in Gag p17, Q65H; found in 9.4% (3/32) of B7 negative participants, and with mutant viruses replicating 11% slower than wild-type in tissue culture. Unlike a previous study in this cohort, infection with viruses carrying mutations in HLA-B*57/58:01 restricted epitopes alone did not impact on viral load setpoint in HLA-mismatched participants, although there was a transient trend towards higher CD4 counts at 3 months post infection (p=0.0551). However, HLA-mismatched participants infected with viruses carrying 3 or more HLA footprints associated with fitness cost in Gag had significantly lower viral load and higher CD4 counts at 3 and 12 months post-infection .These results suggest that multiple mutations generated when viruses are passaged through individuals with beneficial HLAs are needed to attenuate the virus, supporting vaccination methodologies that aim to render the virus less fit.


There is only one study in the literature that found reduced activation of the pacemaker funny current (IF), encoded by the hyperpolarization-activated cyclic nucleotide modulated-4 (HCN4) gene family, in human atrial cells after administration of LPS.Different animal Escherichia Coli; Sigma, St Louis, USA). In addition, 50 pM soluble MD-2 protein was added to the culture medium for enhancing the responsiveness of TLR4 to LPS. Twenty-four hours after LPS addition, electrophysiological recordings were performed at 36°C with the whole-cell patch clamp technique, using an Axopatch 200B amplifier . IF current properties were measured during 6-second hyperpolarizing steps (range -30 to -120 mV), from a holding potential of -30 mV. Voltage control, data acquisition and analysis were accomplished using custom software.Twenty human embryonic kidney (HEK) 293 cells were transfected with Toll-like receptors-4 (TLR4), CD14 and HCN4 cDNAs and after 24 hours were incubated with 1 μg/ml (10 cells) or 10 μg/ml (10 cells) of LPS (from 1/2) values and slope factor (k), derived from voltage-dependent activation curves after Boltzmann fitting to experimental data , than the control cells . IF current densities between -60 and -90 mV weresignificantly higher in untreated cells (0.67 ± 0.5 pA/pF) than in 1 and 10 μg/ml incubated LPS cells .Incubation of cells with both 1 and 10 μg/ml LPS was found to significantly impair IF related to controls, by suppressing the current at membrane potentials between -60 and -90 mV and slowing down current activation. Funny current in LPS-treated cells showed more negative half-maximum activation voltage (VIn conclusion, this study showed in HEK 293 cells a negative impact of LPS upon activation properties of the pacemaker IF current, confirming findings from previous studies on human atrial cells.


Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia. Expression of the Philadelphia chromosome (Ph), i.e. the t9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein, is the hallmark of chronic myeloid leukemia (CML). BCR-ABL1 is not only present in CML patients, but also occurs in 20-30% of acute lymphoblastic leukemia (ALL) cases. Nilotinib AMN107) is an effective secondary generation tyrosine kinase inhibitor (TKI) interacting with the ATP-binding site of BCR-ABL1. Compared to the first generation TKI imatinib, nilotinib not only shows a low IC50 value (IC50 20-60 nM vs. IC50 120-470 nM), but also acts against most imatinib-unresponsive BCR-ABL1 mutation variants 2 chromos, overexp7 is an eIn this study, we set out to dissect the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway to investigate TKI resistance mechanisms and sensitization of Ph+ tumor cells to TKI treatment. Two members of the PI3K/AKT pathway were overexpressed in TKI-resistant cells, GAB2 (Grb-associated binder-2) and MDM2 (human homolog of the murine double minute-2), which stood out as plausible causes for TKI resistance.GAB2 is a critical signal transducer of BCR-ABL1, which couples growth factor and cytokine receptors to downstream effectors, such as PI3K/AKT/mTOR. Persistent phosphorylation of GAB2 Y452, a PI3K recruitment site, confers GAB2-mediated TKI resistance, whereas GAB2 knockdown or haploinsufficiency increases TKI sensitivity . The PI3MDM2 is a downstream effector of PI3K/AKT pathway, stabilized by AKT-dependent phosphorylation . Cancer BEZ235, a dual PI3K/mTOR inhibitor, reduces PI3K and mTOR activity through competitive binding to the ATP-binding site of these enzymes . BEZ235 The cell lines applied in this study are all held by the DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (www.dsmz.de). Detailed references and cultivation protocols have been described previously . NilotinApoptotic cells were detected and quantified with the annexin-V/PI (propidium iodide) method using the TACS Annexin-V-FITC kit  according to the manufacturer’s instructions. Binding of fluorescein isothiocyanate-labeled annexin-V and PI staining of the cells were determined by flow cytometry on the FACSCalibur . For cell cycle analysis cells were fixed with 70% ethanol , washed with phosphate-buffered saline, and stained with PI (20 μg/ml). DNA contents of cells were determined by flow cytometry.5’-GGT CTG TAT CCC GAG AAG C-3’; PI3KCA reverse: 5’-GAG GCC AAT CTT TTA CCA AGC-3’; AKT forward: 5’-GCT CTT TGT GAT GGA TGA GGA-3’; AKT reverse: 5’-CTC AGG CTG CCA TCA TCT G-3’; TSC1 forward: 5’-CTT CTT GCC ATG CTG GAC TC-3’; TSC1 reverse: 5’-CCA CGG TCA GAA TTG AGG TT-3’; TSC2 forward: 5’-CAG GTC TGC AGA GGG TAA AC-3’; TSC2 reverse: 5’-TGC GAT TGT TGA GGC CAC ATT-3’; GβL forward: 5’-GAA TGC CTT GGA GGT CAC AC-3’; GβL reverse: 5’-CCA CAG ACG CGA TGT TCT TG-3’; RAPTOR forward: 5’-TTG TGC CTG AAT GTT GGT GTG-3’; RAPTOR reverse: 5’-CGA TGG TTT CCA GAG CTT TCT-3’; RICTOR forward: 5’-GGC AGC TGA GGC AAA AAC TA-3’; RICTOR reverse: 5’-ACG AAT AAA TGC AGA GAG TAT CAG-3’; mTOR forward: 5’-AGT GGG AAG ATC CTG CAC ATT-3’; mTOR reverse: 5’-TGG AAA CTT CTC TCG GGT CAT-3’; P70S6K forward: 5’-TGA GGA TGA GCT GGA GGA G-3’; P70S6K reverse: 5’-GGC CCT CTG TTC ACA CTA G-3’; MDM2 forward: 5’-TGT TGG TGC ACA AAA AGA CAC TT-3’; MDM2 reverse: 5’-GCA CGC CAA ACA AAT CTC CTA-3’; RPS9 forward: 5’-GGG AAG CGG AGC CAA CAT G-3’; RPS9 reverse: 5’-GTT TGT TCC GGA GCC CAT ACT-3’. Relative expression levels were calculated using the ΔΔ Ct-method.Quantitative PCR was performed on a 7500 Applied Biosystems  real-time PCR system using the manufacturer’s protocol. RNA was prepared using the RNeasy Mini kit . For mRNA quantification, reverse transcription was performed using the SuperScript II reverse transcriptase kit . Expression of genes was assessed using the SYTO-82 PCR Master Mix (Invitrogen) with RPS9 as internal control. Primers were the following: PI3KCA forward: Samples were prepared as described previously . p-mTOR,For RNAi studies, siRNAs  directed against MDM2  and GAB2  were electroporated into cell lines at the final concentration of 20 nM using the EPI-2500 impulse generator . AllStars Neg. Control siRNA (Qiagen) was used as a negative control. Knockdown efficiency was determined by Western blot. Cells were harvested after 24 h or 48 h, respectively, for further studies.t-test. All experiments reported here represent independent duplicate or triplicate replicates. All data are represented as mean value ± SD and significant differences are indicated as *P<0.05, **P<0.01.Data were analyzed using the Student’s Ph+ cell lines JURL-MK2 and SUP-B15 were treated with TKI nilotinib or PI3K/mTOR inhibitor BEZ235 and apoptosis was tested by annexin-V/PI assay. Time-course and dose-response studies showed that JURL-MK2 cells were sensitive to the TKI nilotinib . More thIn addition to BEZ235-induced apoptosis, cell cycle arrest was examined in JURL-MK2 and SUP-B15 cells using PI staining and flow cytometry analysis. Cells were treated with different concentrations of nilotinib or BEZ235. After 24 h, cells were harvested and the percentage of cells in G1 was measured. Both nilotinib and BEZ235 increased the cell fractions in G1 phase in JURL-MK2 cells  left. InJURL-MK2 and SUP-B15 are both Ph+ cell lines, containing unmutated BCR-ABL1 . HoweverConfirming the importance of GAB2 for BCR-ABL1-mediated cell survival, GAB2 knockdown induced apoptosis in JURL-MK2 cells . HoweverSince overexpression of GAB2 was not the reason for nilotinib resistance in SUP-B15, we dissected the downstream signaling chain to find the pathway members responsible for blockage of nilotinib-mediated cell death. To this end, we first compared the mRNA levels of PI3K/AKT pathway factors in JURL-MK2 and SUP-B15 cells by quantitative RT-PCR. Of all relevant genes, only the anti-apoptotic MDM2 showed elevated transcript levels in TKI-resistant SUP-B15 cells . In accoBased on the finding that BEZ235 but not nilotinib reduced MDM2 protein expression in SUP-B15 cells we speculated whether MDM2 overexpression was the cause for nilotinib resistance. To this end, we performed MDM2 knockdown experiments. Suppression of MDM2 alone barely induced apoptosis in the nilotinib-resistant cell line SUP-B15 . HoweverThe BCR-ABL1 oncogene contributes to the development of CML clones. BCR-ABL1 does not only occur in CML, since 20-30% of ALL are also Ph+. TKIs are effective for CML therapy. In a previous study, we tested 19 Ph+ CML and ALL cell lines for TKI responsiveness. 5/19  were TKI-resistant, although none showed mutation in the kinase domain of BCR-ABL1. Resistance was also not independent of BCR-ABL1 size variance. All three BCR-ABL fusion proteins  exhibit deregulated tyrosine kinase activity compared with the native ABL protein . ResistaGAB2 is a scaffold protein, linking plasma membrane receptor signaling (including receptor tyrosine kinases) with downstream effectors. GAB2 acts as signal transducer downstream of BCR-ABL1, thus contributing to TKI resistance in CML cells . GAB2 plWe compared expression of the members of PI3K/AKT signaling pathway and found that of these only MDM2 was overexpressed in the resistant cell line SUP-B15 , consistMDM2 is a protein with a very short turnover time . TherefoWe previously reported that 5/19 Ph+ CML/AML (acute myeloid leukemia) cell lines  were TKI-resistant and that none showed mutations in the kinase domain of BCR-ABL1. We identified the PI3Kα E545G mutation in cell line KCl-22  and the In conclusion, we used Ph+ cell lines JURL-MK2 and SUP-B15 to investigate mechanisms of TKI resistance and sensitization. TKI nilotinib effectively induced cell apoptosis in JURL-MK2, but not in SUP-B15 cells. Inhibition of BCR-ABL1 by nilotinib and simultaneous down-regulation of the anti-apoptotic protein MDM2 by BEZ235 synergized in inducing apoptosis in SUP-B15 cells . BEZ235 Figure S1BEZ235 inhibits phosphorylation of BCR-ABL1 downstream signaling molecule ERK but not STAT5 in SUP-B15 cells. (A) SUP-B15 cells were respectively treated with 100 nM Imatinib, 100 nM nilotinib, 100 nM Everolimus or 2 μM BEZ235 for 24 h. p-ERK and ERK protein levels were analyzed by Western blot. (B) P-STAT5 and STAT5 protein expression in SUP-B15 cells were tested by Western blot after 24 h treatment with different concentrations of nilotinib and BEZ235.(TIF)Click here for additional data file.Figure S2Genomic silencing of PTEN in MHH-TALL-1. (A) Fluorescence in situ hybridization analysis showed intact IKZF1 (left) but loss of the genomic region hosting PTEN (right) covering much of BAC clone RPM1-383D9 (red) and all of the neighboring clone RPMI-879E1. Conventional cytogenetic analysis showed no evidence of genomic loss, hence indicating focal microdeletion of the centromeric part of chromosome band 10q23.31 (not shown). (B) Genomic PCR confirmed the hemizygous loss of PTEN in cell line MHH-TALL1. (C) The remaining PTEN allele had a one base pair insertion leading to a premature stop after amino acid 241, evidenced by sequencing of the RT-PCR product of cell line MHH-TALL1. (D) Cell line MHH-TALL1 did not express the PTEN protein according to Western blot analysis. T, T-cell; B, B-cell; M, myeloid; r, resistant; s, sensitive; n.d., not done.(TIF)Click here for additional data file.


Temperature affects virtually all cellular processes. A quick increase in temperature challenges the cells to undergo a heat shock response to maintain cellular homeostasis. Heat shock factor-1 (HSF-1) functions as a major player in this response as it activates the transcription of genes coding for molecular chaperones  that maintain structural integrity of proteins. However, the mechanisms by which HSF-1 adjusts fundamental cellular processes such as growth, proliferation, differentiation and aging to the ambient temperature remain largely unknown.Caenorhabditis elegans HSF-1 represses the expression of daf-7 encoding a TGF-β (transforming growth factor-beta) ligand, to induce young larvae to enter the dauer stage, a developmentally arrested, non-feeding, highly stress-resistant, long-lived larval form triggered by crowding and starvation. Under favorable conditions, HSF-1 is inhibited by crowding pheromone-sensitive guanylate cyclase/cGMP (cyclic guanosine monophosphate) and systemic nutrient-sensing insulin/IGF-1 (insulin-like growth factor-1) signaling; loss of HSF-1 activity allows DAF-7 to promote reproductive growth. Thus, HSF-1 interconnects the insulin/IGF-1, TGF-β and cGMP neuroendocrine systems to control development and longevity in response to diverse environmental stimuli. Furthermore, HSF-1 upregulates another TGF-β pathway-interacting gene, daf-9/cytochrome P450, thereby fine-tuning the decision between normal growth and dauer formation.We demonstrate here that in Together, these results provide mechanistic insight into how temperature, nutrient availability and population density coordinately influence development, lifespan, behavior and stress response through HSF-1. Caenorhabditis elegans the insulin/IGF-1 and TGF-β signaling pathways co-regulate metabolism, aging, stress tolerance and development . These results suggest that HSF-1 mediates the regulatory effect of DAF-11 on daf7 transcription, and that HSF-1 represses daf7.Under favorable conditions, DAF-7/TGF-β accumulates in two chemosensory neurons, the ASIs, to promote reproductive growth cell non-autonomously . DAF-11/daf11 shares many functions with daf21 encoding an Hsp90 chaperone; daf11 and daf21 have similar epistasis relationships with other genes in the dauer pathway  mutants, too. Expression of daf7 in the daf21(p673); hsf1(sy441) double mutant genetic background was indeed as strong as in the wild-type background  single mutant versus daf-11(−); hsf-1(−) double mutant animals, and found that HSF-1 deficiency strongly suppresses dauer development in daf-11(−) genetic background  mutants animal. Dauer development in daf-21(p673) mutant animals was also blocked by the hsf-1(sy441) mutation   mutation, suggesting that HSF-1 mediates the modulatory effect of temperature on daf-7 transcription. Moreover, hyperactivation of HSF-1 decreased daf-7 transcriptional activity by half at 20°C  in the regulatory region of daf7, 278 base pairs (bp) upstream of the ATG translational initiation site  reporter and a mutated version of this reporter in which 6 critical bases were removed from the canonical HSF-1 binding site (pmutdaf7::gfp)  single mutants and remained strong in daf11(−); hsf1(−) double mutants, expression of the mutant reporter was not significantly changed upon DAF-11 deficiency  mutants as well. Indeed, the long-lived phenotype of daf7(e1372) mutant animals was strongly, but not completely, suppressed by the hsf1(sy441) mutation ; hsf1(sy441) double mutants lived nearly as short as hsf1(sy441) single mutants. This raises the possibility that HSF-1 functions downstream of DAF-7 in ageing control. It is worth to note that the incomplete suppression of longevity in daf7(e1370) mutants by the hsf1(sy441) mutation may result from the fact that sy441 represents a hypomorphic, and not a genetic null, allele.TGF-β signaling influences longevity via the insulin/IGF-1 signaling pathway; loss-of-function mutations in of daf7e172 mutantdaf7(−) null mutant animals correlates with the ambient temperature: the higher the temperature is between 15-25°C, the larger proportion of the population enters the dauer diapause  mutant genetic backgrounds. Since in the temperate and tropical zones the temperature is often above 27°C, nematode populations would frequently exist as non-reproducing dauer larvae under these conditions. This way, HSF-1 regulates both a dauer-inhibiting (daf7) and a dauer-inducing (daf9) TGF-β pathway component in order to allow the populations to survive (some dauer larvae) and propagate (many reproductive adults) simultaneously under environmental stress, thereby maximizing the chance of the population to survive.HSF-1 stimulates y Figure D. daf7 rer stage . WithoutC. elegans development. DAF-11 is a receptor guanylate cyclase that is implicated in chemosensation and behavior , VC3071 hsf-1(ok600)/hIn1[unc-101(sy241)]I, CF1824 muEx265[hsf-1p::hsf-1cDNA + myo-3::GFP], TTV200 bjIs10[hsf-1p::hsf-1cDNA + myo-3::GFP] backcrossed 4x, TTV201 hsf-2(tm4607)I backcrossed 4x, TTV116 bjEx1[pdaf-7::daf-7::gfp + unc-119(+)], TTV117 bjEx9[pmutdaf-7::daf-7::gfp + unc-119(+)].The wild-type strain corresponds to var. Bristol (N2). The following mutant and transgenic strains were used in this study: PS3551 daf-7::gfp reporter, a genomic fragment containing 3,8 kb of the 5’ regulatory region and the first exon of daf-7 was amplified by the following forward and reverse primers: 5’-aaa tcc gag tcc gtg aaa tg-3’ and 5’-aaa cac cgg gag tga aga tg-3’. The resulting fragment was cloned into pGEM®-T Easy vector and subcloned into the vector pPD95.75, using the SphI and SalI restriction enzymes. QuikChange® II Site-Directed Mutagenesis Kit (Stratagene) was used to generate the mutant version of this reporter, with the primers: 5'-caa ttc cgc aaa att ttc tgg ctt ttc tct acg gta tag atg-3' and 5'-cat cta tac cgt aga gaa aag cca gaa aat ttt gcg gaa ttg-3’. These daf-7 genomic fragments were inserted in frame with gfp in these constructs. Transgenic strains were generated by biolistic transformation using Biolistic PDS-1000/He particle delivery system (BioRad). 10-15μg linearized plasmid DNA was bombarded onto unc-119(ed3) mutant L4/adult hermaphrodites. Fluorescent images were taken by an Olympus BX51 epifluorescence microscope.To generate a pMeasurements were performed by the Image J software. Briefly, images were captured with Olympus BX51 epifluorescence microscope with a given exposure time. Cells of interest were selected, and mean grey values were measured. Fluorescent intensity of selected area was calculated by subtracting mean grey value of the background from mean grey value of the object of interest (the same size of areas was selected).E. coli OP50 to lay eggs for 4–5 hours, and then removed. F1 young adults were transferred to NGM plates supplemented with 300μg/μl FUDR (5-fluoro-2'-deoxyuridine). Animals were considered dead when they stopped pharyngeal pumping and responding to touching. SPSS software was used to calculate mean life span and perform statistical analysis. P values for comparing Kaplan-Meyer survival curves between two groups were determined using log-rank (Mantel-Cox) tests.Life span assays were carried out at 25°C, as described previously . Brieflypmp-3), relative to the wild-type strain. Primers for the housekeeping gene pmp-3: 5’-gtt ccc gtg ttc atc act cat-3’and 5’-aca ccg tcg aga agc tgt aga-3’, and for daf-7: 5’-caa caa tgt gat agg caa cga-3’and 5’-aac tac gca cgc aca gac ac-3’. The results represent the mean of 3 independent experiments. To compare the means of dauer progeny, variables were analyzed by Student’s t-test. Results are expressed as mean ± S.E.M.RNA samples were extracted from 100–200 synchronized L1 larvae, using a PureLink™ Micro-to-Midi Total RNA Purification System (Invitrogene). RNase-free DNase I (Fermentas) was used to eliminate genomic DNA contamination. DNA-free RNA samples were then converted to cDNA, using random hexamers by RevertAid First Strand cDNA Synthesis Kit. Amplification reactions were carried out in a total volume of 20 μl, using LightCycler® FastStart DNA Master SYBR Green I. The reaction conditions for the LightCycler® 2.0 Instrument (Roche) were: 95°C for 5 min, followed by 45 cycles of 95°C for 10s, 60°C for 5s, 72°C for 10s. Fluorescence was measured at each cycle at 72°C. Melting curve analysis was performed after PCR to assess the presence of a unique final product. Gene expression data are presented as the fold change in mRNA transcript abundance in mutant strains, normalized to one endogenous reference gene , and percentage of dauer larvae was scored typically after 72, 60 and 44 hours, respectively. At least three plates per strain were assayed. Results are expressed as mean ± S.E.M. Data were analyzed by one-way ANOVA and hsf-1 cDNA clone  was cut by HindIII and KpnI, and the resulting 1333bp-long cDNA fragment was cloned into the L4440 “feeding” vector. The construct was then transformed into the bacterial strain Escherichia coli HT115(DE3).An The authors declare that they have no competing interests.JB, KTV and TV designed research; JB, AP, MK, BH and KTV performed research; JB, KTV and TV analyzed data; JB and TV wrote the paper. All authors read and approved the final manuscript.


To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions.  Notch signaling pathway is an evolutionarily conserved signal transduction cascade in flies, worms and vertebrates. It is a short-range cell communication. Through the Notch signaling pathway, signal-sending cells transfer a lateral inhibitory signal to the adjacent signal-receiving cells to control cell fate decision during development. It also plays roles in cell proliferation, cell death and self-renewal of adult stem cells Drosophila and Lag-2 for C. elegans) on the surface of signal-sending cells with the Notch receptor on signal-receiving cells. Accompanying the interaction, ligands are internalized into cells that provide a pulling force on the Notch receptors leading to the exposure of the second cleavage site cut by ADAM metalloproteases. Then, the third cleavage of the Notch receptor by γ-secretase releases the Notch intracellular domain (NICD) into the nucleus. NICD forms a complex with CSL transcription factors  to regulate the expression of downstream genes Notch signaling is activated by the interaction of DSL ligands . Mind bomb is an E3 ubiquitin ligase with two mib/herc2 domains, zz zinc finger domain, two mib repeats, eight ankyrin repeat domains (AND) and three RING finger domains . Mib in Ubiquitylation is a posttranslational modification with ubiquitin by the sequential action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin protein ligase (E3). Ubiquitin can be conjugated to substrates at many different sites and in many distinct topological configurations, which behaves as complex signals to regulate protein activity, localization and degradation Studies have shown that both HIF activation and deregulated Notch signaling were linked to tumorigenesis and development The most well-known function of Mind bomb is its role in the Notch signaling pathway. However, it is not the only function of Mib. Mib has been reported to be involved in the apoptosis by interaction with death-associated protein kinase (DAPK) and cellular Fas-associated death domain (FADD)-like IL-1b converting enzyme (FLICE)-like inhibitory proteins (cFLIP) In this study, to further explore the function of Mib and Mib2, yeast two-hybrid screens were used to identify Mib/Mib2-interacting proteins. By this approach we isolated 81 putative Mib/Mib2-binding proteins and classified them into six groups: ubiquitin proteasome system, cytoskeleton, trafficking, replication/transcription/translation, cell signaling and others. The potential roles of Mib and Notch activity in these pathways require further investigation. Additional analysis was carried out on several interesting candidates. Hif1an/Fih-1  and Trabid/Zranb1  were shown to interact with and be ubiquitylated by Mib/Mib2 in COS7 cells. Usp1 and Usp9 can be co-immunoprecipitated by Mib/Mib2. These data suggest that Mib and Mib2 may be required for hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway in addition to their prominent function in Notch signaling.To further investigate Mib function and identify putative Mib/Mib2-binding proteins, two yeast two-hybrid screens with different interaction strategies were performed using different regions of Mib and Mib2 as baits  to screeIn the second screen with CytoTrap system, the bait and prey proteins are expressed and retained in the cytoplasm, where, unlike in the nucleus, they may undergo post-translational modifications. In addition, transcriptional activators and inhibitors may be used as baits to screen for protein-protein interactions. In this screen, we picked up a total of 95 positive clones that have in-frame cDNA sequence with the myristylation sequence, in which 52 clones of 28 genes by Mib-M-Ank (454–785 aa), 6 clones of 4 genes by Mib-C-RF123b (789–1030 aa) and 37 clones of 20 genes by Mib-ZnF+Ank (74–785 aa) . Mib-M-AIn these two screens, the baits Mib-N 1–462 aa), Mib-N+Ank (1–782 aa) and Mib2-N+Ank (54–780 aa) showed autoactivation and were not used for screens to avoid false positive clones. Interestingly, all of these autoactivated baits contains two mib/herc2 domains, whose configuration and/or conformation may have unexpected higher affinity with or non-specific binding to the reporter constructs or non-binding activation of Ras signal. In the comparison of these two screens, many positive clones are functionally related and can be classified into six groups: ubiquitin proteasome system, cytoskeleton, trafficking, replication/transcription/translation, cell signaling and others  and 2. A aa, Mib-ribosomal protein S27a (rps27a)/ubiquitin A-80-residue ribosomal protein fusion product (UBA80) (46), ubiquitin C (ubc) (45), ubiquitin B (ubb) (31), ubiquitin specific protease 5 (usp5) (31), ubiquitin A-52 residue ribosomal protein fusion product (uba52) (30), myosin, light chain, phosphorylatable, fast skeletal muscle a (mylpfa) (21), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide b (ywhaz) (14-3-3 zeta) (15), actinin alpha 4 (actn4) (7), insulin-degrading enzyme (ide) (7), nascent polypeptide-associated complex alpha polypeptide (naca) (7), DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (ddx5) (6). They belong to either proteasome/ubiquitin system or structural housekeeping genes, except 14-3-3 zeta, ide, naca and ddx5. The higher number of clones may reflect the abundance of these genes.Some genes are frequently picked-up. Those that have more than 5 clones identified include In our screens, the ubiquitin proteins: ubiquitin B, ubiquitin C, ubiquitin A-52 and ubiquitin A80 appeared with a high frequency. Proteasome subunits including proteasome 26S subunit 1 (psmd1), proteasome subunit 5 (psma5) and proteasome 26S subunit 4 (psmd4) were also identified from both screens, reflecting a role of protein degradation and the E3 ligase activity of Mib and Mib2. Other intriguing interacting proteins were deubiquitylases (DUBs): ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 5 (Usp5), ubiquitin specific protease 9 (Usp9) and tumor-necrosis-factor-receptor-associated factor-binding domain (Trabid) in the ubiquitin proteasome system group  and 2.mib or -mib2 and Myc- or HA- tagged DUBs. The whole cell lysate was immunoprecipitated (IP) by anti-FLAG antibody and then subjected to Western blotting (IB) by anti-Myc antibody or anti-HA antibody to detect the interacting proteins. The results indicated that Usp1 C (from 349 to 772 amino acids), Usp9 and Trabid C (from 295 to 716 amino acids) were co-immunoprecipitated with Mib , heat shock cognate (Hsc70) and factor inhibiting HIF-1 (Fih-1) were investigated to verify their binding activities with Mib and Mib2 for the reasons that they have been shown to interact with Notch signaling genetically and/or biochemically ubiquitin, different pCS2-FLAG-mib or -mib2 and pCS2-MT-fih-1 or -trabid C (295–716 aa). The whole cell lysate was immunoprecipitated with anti-Myc antibody and then subjected to immunoblotting with anti-Myc antibody as a control or anti-HA to detect the ubiquitylated proteins. Both Mib and Mib2 facilitated Fih-1 and Trabid C ubiquitylation -α and interleukin (IL)-1 are pro-inflammatory cytokines that initiate signaling pathways in endothelial cells leading to activation of nuclear factor κB (NF-κB) and plays an important role in the regulation of immune and inflammatory responses 2 sensing enzymes, such as HIF-α-specific prolyl hydroxylase domain proteins (PHDs) and Hif1an/Fih-1, an asparaginyl hydroxylase. FIH-1 interacts with and mediates hydroxylation of HIF-1α and, therefore, inhibits HIF-1 transcriptional activity The regulation of hypoxia signaling pathway is conducted by ORecently, Fih-1 was found that it can associate with and hydroxylate proteins containing AND, such as Notch receptor 1, 2 and 3 Despite the fact that Mib was not identified in a previous proteomics-based screen for FIH substrates Drosophila indicates that Fat facets (Usp9 ortholog) promotes endocytosis of Delta by deubiquitylating Liquid facets (Epsin ortholog) Enodcytosis of ligands is required for the activation of Notch signaling Mib may also participate in other trafficking processes. Trafficking molecules like exocyst complex component 1, exocyst complex component 3, and vesicle docking protein p115 were found in our screens. Similarly, other trafficking components have also been identified by other group, such as Importin alpha and beta, Kinesin heavy chain member 2, Nipsnap1, Rba11-F1p2, Rab11-FIP5 and early endosome antigen 1 It has been reported that yeast protein interactomes obtained from different research groups by large-scale two-hybrid screens fail to overlap to a great extent Drosophila indicated that Mib2 expressed in founder myoblasts regulates myoblast fusion and muscle stability through a Notch-independent way Drosophila myogenesis, the putative Mib-interacting proteins related to cytoskeleton do implicate such a role in vertebrates.In our screens, several proteins involved in the cytoskeleton, including actinin alpha 2, actinin alpha 3, actinin alpha 4, myosin light chain and cofilin 1, were identified. Cytoskeleton member like dystrobrevin and dystrophin-associated protein A1 were also identified as Mib-interacting proteins by affinity purification and mass spectrometry Proteins involved in cell signaling also appear in our screens, such as polo-like-kinase 4, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteins (14-3-3), TGF-beta activated kinase 1/MAP3K7 binding protein 3 (TAK1-binding protein 3), creatine kinase, and casein kinase 2 beta (ck2b), suggesting that Mib/Mib2 are potentially involved in several signaling pathways in addition to the Notch signaling pathway. Indeed, Mib plays a positive role in the Wnt signaling pathway by regulating the stability and localization of RYK mib and mib2Danio rerio embryo (stages 18–20 hpf) RP1 cDNA library (Hybrigenics) constructed into pP6 plasmid for C-terminal Gal4 transcription activating domain fusion proteins. Mib-N+Ank (1–782 aa) and Mib2-N+Ank (54–780 aa) have autoactivation effect and, therefore, are not used for further screen. 60 million colonies for Mib-C-RF123a (783–1029 aa) and 52 million colonies for Mib2-C-RF12 (781–999 aa) were respectively screened. cDNA fragments corresponding to positive “prey” clones were amplified by PCR and sequenced at their 5′ and 3′ junctions. The resulting sequences were searched against GenBank using a fully automated procedure, assigned a quality score  indicative of the confidence of interaction For the first yeast two-hybrid screen, a conventional LexA and Gal4 system relying on transcriptional activation of reporter genes in the nucleus to detect interactions was used. Bait fragments used for this screen were Mib-N+Ank (1–782 aa), Mib-C-RF123a (783–1029 aa), Mib2-N+Ank (54–780 aa) and Mib2-C-RF12 (781–999 aa) . Yeast tmib cDNA sequences (using BamHI and XhoI as cloning sites). These baits were then used to screen the prey, pMyr-cDNA library  within the temperature-sensitive yeast mutant strain cdc25H according to the manufacturer's instructions (Stratagene). cdc25 is the yeast homolog of the human Sos (hSos) gene, which encodes a guanyl nucleotide exchange factor that activates Ras signal transduction pathway. The cdc25 mutation present in the cdc25H strain allows normal growth at the permissive temperature (25°C), but prevents growth at 37°C. pSos bait vector generates a fusion protein of hSos and the bait protein. pMyr-cDNA is expressed as a fusion protein with a myristylation sequence that anchors the fusion protein to the plasma membrane. The physical interaction of the bait and prey proteins results in the localization of hSos to the plasma membrane and reconstitutes Ras-signaling pathway activity, which allows the yeast growth at both 37°C and 25°C. pSos-Mib-N (1–462 aa) was unsuitable for detecting protein-protein interactions in the CytoTrap system, because the bait plasmid cotransformed with the pMyr empty vector autoactivated Ras signal and induced cdc25H yeast growth at 37°C. The screen then focused on the other three baits. Around 0.4, 0.5 and 0.5 million colonies were screened for Mib-M-Ank (454–785 aa), Mib-C-RF123b (789–1030 aa) and Mib-ZnF+Ank (74–785 aa), respectively. Plasmid DNA was isolated from the putative positive pMyr-cDNA clones. The two-hybrid interaction was confirmed by retransformation of the yeast strain cdc25H with the bait vector and purified prey plasmid. All positive prey plasmids were sent for DNA sequencing by the 5′ primer (5′-ACTACTAGCAGCTGTAATAC-3′) in the pMyr vector and blasted again NCBI database. Clones that have in-frame cDNA sequence with the myristylation sequence were finally selected and listed in For the second yeast two-hybrid screen, zebrafish Mib-N (1–462 aa), Mib-M-Ank (454–785 aa), Mib-C-RF123b (789–1030 aa) and Mib-ZnF+Ank (74–785 aa)  were submib and -mib2 were made by subcloning the PCR-amplified fragments into the pCS2+FLAG vector fih-1 and partial trabid C (295–716 aa) cDNA fragments were PCR-amplified and subcloned into pCS2-MT vector at EcoRI and XhoI sites. Full-length trabid cDNA fragments were PCR-amplified and subcloned into pCS2-MT vector at XhoI and XbaI sites. C-terminal usp1 C (349–772 aa) cDNA fragments were PCR-amplified and subcloned into pCS2-MT vector at EcoRI and XhoI sites. Full-length usp9 cDNA fragment was PCR-amplified and subcloned into pCMV-HA vector at SfiI and NotI sites. All PCR products were verified by DNA sequencing. pcDNA3.1-HA-ubiquitin is a gift from Dr. Margaret A. Shipp. Usp1, Usp9, Trabid and Fih-1 protein sequences are shown in Supporting Information with comparison to those of other species (pCS2-FLAG- species –S4.fih-1 was cotransfected with 4 µg of pCS2-FLAG-mib or -mib2 into COS7 cells. 2 µg of pCS2-MT-trabid C (295–716 aa), pCS2-MT-usp1 (349–772 aa) or pCMV-HA-usp9 was cotransfected with 2 µg of pCS2-FLAG-mib or -mib2 into COS7 cells. Cells were harvested and lysed 24 h after transfection in IONIC buffer . The cell lysate was clarified by centrifugation and incubated with anti-FLAG M2 affinity gel (Sigma) for 2 h at 4°C. The gel was boiled in SDS gel loading buffer and the eluted proteins were electrophoresed on a SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore). Blots were incubated with primary antibody: anti-HA (Protech), anti-Myc (Santa Cruz) or anti-FLAG (Sigma) for 2 hr. The signal was visualized using a secondary antibody: HRP-conjugated anti-mouse IgG antibody (GeneTex) or HRP-conjugated anti-rabbit IgG antibody (GeneTex), with a chemiluminescence detection system (PerkinElmer Inc).COS7 cells ubiquitin, 2 µg of pCS2-MT-fih-1 [or pCS2-MT-trabid C (295–716 aa)] as substrate and 4 µg of pCS2-FLAG-mib or -mib2 as putative E3 ubiquitin ligases. 2 µM proteasome inhibitor MG132 was added to the cell culture medium after 24 h. Cells were harvested at 48 h and lysed in IONIC buffer and 10 µM MG132. The cell lysate was clarified by centrifugation and incubated with rabbit anti-Myc antibody (Santa Cruz) for 2 h at 4°C, and then incubated with protein A Sepharose beads for 1 h at 4°C. The eluted proteins were electrophoresed on a SDS-PAGE followed by Western blotting with mouse anti-HA (Roche) or mouse anti-Myc (Roche) antibody.COS7 cells were transiently transfected with 2 µg of pcDNA3.1-HA-Figure S1Alignment of Usp1 amino acid sequences. Alignment of zebrafish Usp1 , chicken Usp1 , mouse Usp1  and human Usp1 . Zebrafish Usp1 bears 51.49%, 52.39% and 53.14% amino acids identity to its counterparts of chicken, mouse and human, respectively. Red line indicates the region of ubiquitin-specific protease (USP) domain.(PDF)Click here for additional data file.Figure S2Alignment of Usp9 amino acid sequences. Alignment of zebrafish Usp9 , human Usp9  and chicken Usp9 . Zebrafish Usp9 bears 90.04% and 92.58% amino acids identity to its counterparts of human and chicken, respectively. Red line indicates the region of ubiquitin-specific protease (USP) domain.(PDF)Click here for additional data file.Figure S3Alignment of Trabid amino acid sequences. Alignment of zebrafish Trabid , African clawed frog Trabid , human Trabid  and mouse Trabid . Zebrafish Trabid bears 78.88%, 80.17% and 80.31% amino acids identity to its counterparts of Xenopus, human and mouse, respectively. Blue line indicates the region of Npl4 zinc finger domain (NZF). Green line indicates the region of ankyrin repeat. Red line indicates the region of ovarian tumor protease (OTU) domain.(PDF)Click here for additional data file.Figure S4Alignment of Fih-1 amino acid sequences. Alignment of zebrafish Fih-1 , human Fih-1  and mouse Fih-1 . Zebrafish Fih-1 bears 81.76% and 82.35% amino acids identity to its counterparts of human and mouse, respectively. Purple line indicates the region of Jmjc domain.(PDF)Click here for additional data file.


Herpes simplex virus type 1 (HSV-1) has a complicated life-cycle, and its genome encodes many components that can modify the cellular environment to facilitate efficient viral replication. The protein UL14 is likely involved in viral maturation and egress , and it facilitates the nuclear translocation of viral capsids and the tegument protein VP16 during the immediate-early phase of infection . UL14 of herpes simplex virus type 2 exhibits multiple functions .To better understand the function(s) of UL14, we generated VP16-GFP-incorporated UL14-mutant viruses with either single (K51M) or triple  amino acid substitutions in the heat shock protein (HSP)-like sequence of UL14. We observed the morphology of cells infected with UL14-null virus and amino acid-substituted UL14-mutant viruses at different time points after infection.UL14(3P)-VP16GFP and UL14D-VP16GFP (UL14-null) viruses caused similar defects with respect to growth kinetics, compartmentalization of tegument proteins, and cellular morphology in the late phase. Both the UL14D-VP16GFP and UL14(3P)-VP16GFP viruses led to the formation of an aggresome that incorporated some tegument proteins but did not include nuclear-egressed viral capsids.Our findings suggest that a cluster of charged residues within the HSP-like sequence of UL14 is important for the molecular chaperone-like functions of UL14, and this activity is required for the acquisition of functionality of VP16 and UL46. In addition, UL14 likely contributes to maintaining cellular homeostasis following infection, including cytoskeletal organization. However, direct interactions between UL14 and VP16, UL46, or other cellular or viral proteins remain unclear. O'Hare. Viral stocks were prepared in Vero or rabbit skin cells, and were subsequently titrated separately on Vero cell monolayers. Growth curves were obtained as previously reported [® 546-conjugated phalloidin , mouse monoclonal anti-α-tubulin , mouse monoclonal anti-vimentin clone V9 , rabbit polyclonal anti-proteasome 20S α5 , mouse monoclonal anti-Hsp70 , Alexa Fluor® 555-conjugated goat anti-mouse IgG1, Alexa Fluor® 647-conjugated goat anti-mouse IgG1, Alexa Fluor® 555-conjugated goat anti-rabbit IgG, and Alexa Fluor® 647-conjugated goat anti-rabbit IgG.The following commercial antibodies were used: mouse monoclonal anti-HSV1+HSV2 ICP5 , mouse monoclonal anti-HSV1 ICP8 , Alexa Fluor® Alexa Fluor® labeling kit (Invitrogen). Samples were examined under a Zeiss LSM510 confocal immunofluorescence microscope.Cells grown on coverslips were washed with phosphate-buffered saline (PBS) and fixed for 10 min in 4% paraformaldehyde in PBS at room temperature. For GFP visualization, the coverslips were mounted directly onto glass slides with PermaFluor (Thermo). For indirect immunofluorescence, fixed cells were permeabilized in 0.1% Triton X-100 in PBS for 5 min at room temperature. Each coverslip was inverted onto a droplet (20 ml) of blocking buffer  on a clean Parafilm sheet for 30 min at room temperature. Primary and secondary antibodies  were diluted in blocking buffer and reacted for 30 min at room temperature. To stain with two mouse primary antibodies at once, we used the Zenon2. The culture medium was then replaced with newly prepared medium containing 2% fetal bovine serum. Cells and supernatants were harvested at the indicated times after infection. Virus progeny were titrated on Vero cells by plaque assays.Analysis of one-step growth kinetics was performed as described  HEp-2 ceGFP: green fluorescence protein; h.p.i.: hours post-infection; HSP: heat shock protein; HSV: herpes simplex virus; IE: immediate-early; MOI: multiplicity of infection; PBS: phosphate buffered saline; PQC: protein quality control; UL14D: UL14-deleted; UL14(3P): three-point mutated  UL14; VICE: virus induced chaperone enriched.The authors declare that they have no competing interests.AO, YY, and YN designed the research, AO, YY, and YM performed the experimental work, AO conducted the data analysis and drafted the manuscript, and YY, YM, and HK participated in the data analysis and review of the manuscript. All authors read and approved the final manuscript.


Systemic-to-pulmonary collateral flow (SPCF) may constitute a risk factor for increased morbidity and mortality in patients with single-ventricle physiology (SV). However, clinical research is limited by the complexity of multi-site two-dimensional (2D) cardiovascular magnetic resonance (CMR) flow assessment. We sought to validate four-dimensional flow (4D-flow) for concise quantification of SPCF in patients with SV.29 patients with SV physiology prospectively underwent CMR (1.5T) to quantify SPCF  and 20 healthy volunteers  served as controls. Five 2D-flow measurements  were performed and SPCF  was calculated and compared with 4D-flow measurements and calculations. Additionally, 4D-flow measurements were used to calculate SPCF as pulmonary venous minus pulmonary arterial flow.The comparison between 4D-flow and 2D-flow showed good Bland-Altman agreement for all individual vessels , calculated SPCF (-0.02±0.18 l/min/m2), low intra and inter-observer variance (ICC>0.95[0.91-0.97]) and significantly shorter 4D-flow acquisition-time . 4D-flow in patients versus controls revealed (1) good agreement between systemic versus pulmonary estimator for SPFC; (2) significant SPCF in patients  and not in controls (0.01+0.16 l/min/m2) and (3) inverse relation of right/left pulmonary artery perfusion and right/left SPCF .4D-flow is reliable, operator-independent and more time-efficient than 2D-flow to quantify SPCF. There is considerable SPCF in BCPC and Fontan patients. SPCF was more pronounced towards the respective lung with less pulmonary arterial flow suggesting more collateral flow where less anterograde branch pulmonary artery perfusion.


Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWCON and AWCOFF, by inhibiting a calcium-mediated signaling pathway in the future AWCON cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWCON fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWCON cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWCON identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWCON neurons. tir-1 expression is downregulated through its 3′ UTR in AWCON, in which mir-71 is expressed at a higher level than in AWCOFF. In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3′ UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWCON identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.The  C. elegans, a specific sensory neuron pair communicates to establish stochastic asymmetric identities by inhibiting a calcium signaling pathway in the neuron that becomes an induced identity. However, it is not understood how cell–cell communication inhibits the calcium signaling pathway in the induced neuronal identity. In this study, we identify a microRNA that represses the expression of a key molecule in the calcium signaling pathway to promote the induced neuronal identity. Overexpression of the microRNA causes both neurons of the pair to become the induced identity, similar to the mutants that lose function in the calcium signaling pathway. In addition, the stability of the mature microRNA is dependent on a claudin-like protein and a gap junction protein, the two parallel signals that mediate communication of the neuron pair to promote the induced neuronal identity. Our results provide insight into the mechanism by which cell–cell communication inhibits calcium signaling to establish stochastic asymmetric neuronal differentiation.Cell identity determination requires a competition between the induction of cell type–specific genes and the suppression of genes that promote an alternative cell type. In the nematode  C. elegansDrosophila eye, the kinase Warts and PH-domain containing Melted repress each other's transcription in a bistable feedback loop to regulate the two alternative R8 photoreceptor subtypes expressing Rhodopsin Rh5 or Rh6 C. elegans sensory system, two sets of transcription factors and miRNAs reciprocally repress each other to achieve and stabilize one of the two mutually exclusive ASEL and ASER taste neuronal fates Cell fate determination during development requires both the induction of cell type specific genes and the suppression of genes that promote an alternative cell fate C. elegans left and right sides of Amphid Wing Cell C (AWC) olfactory neurons specify asymmetric subtypes through a novel mechanism independent of the Notch pathway in late embryogenesis ON neuron expresses the chemoreceptor gene str-2 and the contralateral AWCOFF neuron does not str-2 on the left and the other 50% express it on the right. Ablation of either AWC neuron causes the remaining AWC neuron to become AWCOFF, suggesting that AWCOFF is the default state and the induction of AWCON requires an interaction or competition between the AWC neurons ON state. These results suggest that the synapses could mediate the AWC interaction for asymmetry The nsy-4, encoding a claudin-like tight junction protein, and nsy-5, encoding an innexin gap junction protein, act in parallel to downregulate the calcium-mediated UNC-43 (CaMKII)/TIR-1 (Sarm1)/NSY-1 (MAPKKK) signaling pathway in the future AWCON cell ON and AWCOFF identities are maintained by cGMP signaling, dauer pheromone signaling, and transcriptional repressors unc-43(CaMKII), tir-1 (Sarm1), and nsy-1 (MAPKKK) are also implicated in the maintenance of AWC asymmetry in the first larval (L1) stage nsy-4 and nsy-5 in the AWCON cell.OFF identity tir-1 expression may represent an efficient mechanism to inhibit calcium signaling in the cell becoming AWCON. In support of this idea, a prior large scale examination of potential miRNA targets indicated that tir-1 and unc-43 may be downregulated by this class of RNAs mir-71 in stochastic AWC asymmetry by characterizing its role in downregulation of the calcium signaling pathway in the AWCON cell. We show that mir-71 acts downstream of nsy-4/claudin and nsy-5/innexin to promote AWCON in a cell autonomous manner through inhibiting tir-1 expression, in parallel with other processes. We also show that nsy-4 and nsy-5 are required for the stability of mature mir-71. Our results suggest a mechanism for genetic control of AWC asymmetry by nsy-4 and nsy-5 through mir-71-mediated downregulation of calcium signaling.The TIR-1/Sarm1 adaptor protein assembles a calcium-signaling complex, UNC-43 (CaMKII)/TIR-1/NSY-1 (ASK1 MAPKKK), at AWC synapses to regulate the default AWCON gene str-2 in the default AWCOFF cell ON cell. miRNAs are small non-coding RNAs that are robust in mediating post-transcriptional and/or translational downregulation of target genes C. elegans, miRNAs are processed from premature form into mature form by alg-1/alg-2 (encoding the Argonaute proteins) and dcr-1 (encoding the ribonuclease III enzyme Dicer) unc-43 and tir-1 in dcr-1 mutants unc-43 and tir-1 may be downregulated by miRNAs. Thus, we hypothesized that miRNAs may play a role in downregulation of the UNC-43/TIR-1/NSY-1 signaling pathway in the cell becoming AWCON.The calcium-regulated UNC-43 (CaMKII)/TIR-1 (Sarm1)/NSY-1 (ASK1 MAPKKK) signaling pathway suppresses expression of the AWCunc-2, unc-36, egl-19, unc-43, tir-1, nsy-1, and sek-1, in the AWC calcium signaling pathway. Only the miRNAs that fit the following criteria were selected for further analysis: 1) At least 6 nucleotides in the seed region (position 1–7 or 2–8 at the 5′ end) of a miRNA is perfectly matched to the target 3′ UTR; 2) The seed match between a miRNA and its target 3′ UTR is conserved between C. elegans and a closely related nematode species C. briggsae, since evolutionary conservation between C. elegans and C. briggsae genomes is useful in identifying functionally relevant DNA sequences such as regulatory regions http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/) http://www.targetscan.org/worm_12/) mir-71, mir-72, mir-74, mir-228, mir-248, mir-255) predicted to target unc-2, unc-43, tir-1, nsy-1, and sek-1 (http://pictar.mdc-berlin.de/) http://146.189.76.171/query.php) To test this hypothesis, we took a computational approach to identify miRNAs predicted to target the 3′ UTRs of known genes, including nd sek-1 . A subseodr-3 promoter, expressed strongly in AWC neuron pair and weakly in AWB neuron pair str-2p::GFP (AWCON marker) expression in only one of the two AWC neurons  led to str-2p::GFP expression in both AWC neurons (2AWCON phenotype) (ON phenotype. We found that mir-71(OE) animals overexpressing mir-71, predicted to target tir-1 and nsy-1, had a strong 2AWCON phenotype    [12], . Wep<0.001) . These rmir-71 in AWC asymmetry, we analyzed str-2p::GFP expression in the mir-71(n4115) deletion null allele mir-71(n4115) mutants displayed wild-type AWC asymmetry ; mir-248(OE) animals than in mir-71(OE) (data not shown). These results suggest that mir-71 may not act redundantly with mir-248 to regulate tir-1 expression in AWC asymmetry. To knockdown mir-248 expression, we made an anti-mir248 transgene expressing short hairpin RNA (shRNA), consisting of both sense and antisense sequences of mir-248, in AWC. The anti-mir-248 transgene caused an AWC phenotype similar to mir-248(OE) (data not shown), suggesting that the effect of the anti-mir-248 transgene on AWC asymmetry is not through knockdown of mir-248 but mainly due to overexpression of sense mir-248 in the shRNA construct.To further determine the requirement of symmetry , suggestprograms . mir-71  2AWCOFF . To testDrosophila eye mir-71, we crossed mir-71(n4115) into sensitized backgrounds including tir-1(ky388), nsy-4(ky616), and unc-76(e911) mutants. tir-1(ky388) is a temperature-sensitive (ts) allele that caused a 2AWCON phenotype in 29% of animals at 15°C (ON phenotype of tir-1(ky388ts) mutants was significantly suppressed by mir-71(n4115), such that 20% of mir-71(n4115); tir-1(ky388ts) double mutants had a 2AWCON phenotype  gene, encoding a protein phosphatase 2A regulatory subunit (ppfr-1 in mir-71(n4115) mutants may affect ppfr-1 activity leading to suppression of the tir-1(ky388ts) 2AWCON phenotype. To test this possibility, we analyzed AWC phenotypes in ppfr-1(tm2180); tir-1(ky388ts) double mutants. ppfr-1(tm2180) has a 1027 bp deletion removing the first three exons and therefore is a potential null allele (ON phenotype of ppfr-1(tm2180); tir-1(ky388ts) double mutants was not significantly different from that of tir-1(ky388ts) single mutants (ppfr-1 is not required for AWC asymmetry and that suppression of the tir-1(ky388ts) 2AWCON phenotype was most likely caused by loss of mir-71 activity in mir-71(n4115) mutants. subunit . It is pl allele  [43]. Th mutants . This rensy-4 claudin-like gene and the unc-76 axon guidance pathway gene induce the AWCON state by inhibiting the downstream calcium-signaling pathway. Loss-of-function mutations in nsy-4 and unc-76 cause a partially penetrant 2AWCOFF phenotype (mir-71(n4115) mutations significantly enhanced the 2AWCOFF phenotype of nsy-4(ky616) and unc-76(e911) mutants (p<0.001). On the other hand, the 2 AWCON phenotype of nsy-4(OE) trasnsgenic animals overexpressing nsy-4 in AWCs was significantly suppressed in nsy-4(OE); mir-71(n4115) double mutants  animals. The GFP intensity of each sensor construct in an individual AWC neuron was normalized to the nucleus-localized TagRFP intensity of the transgene odr-3p::2Xnls-TagRFP::unc-54 3′ UTR in the same cell. The unc-54 3′ UTR does not contain any strongly predicted mir-71 sites. The normalized GFP intensity of each sensor construct was compared between mir-71(OE) animals and their siblings losing the mir-71(OE) transgene in the L1 stage, during which tir-1 is functional for the maintenance of AWC asymmetry mir-71(OE) animals, compared with wild type, had a significantly reduced normalized expression level of GFP from the tir-1 3′ UTR sensor construct  animals  mutants  animals compared with wild type  animals (tir-1 5′ UTR does not affect mir-71(OE)-mediated suppression of gene expression through the tir-1 3′ UTR.Interactions between the 5′ and 3′ UTRs have been shown to regulate translation in mammalian cells nstructs . Similar1 5′ UTR , the nor p<0.04; . However animals . These rnsy-1 3′ UTR was also predicted to contain a mir-71 binding site by the four programs used in this study (nsy-1 3′ UTR was not significantly different between wild-type and mir-71(OE) animals  animals overexpressing tir-1 in AWC had a 2AWCOFF phenotype tir-1(OE) 2AWCOFF phenotype as readout to determine if mir-71 acts through the tir-1 3′ UTR to suppress the AWCOFF fate. We made tir-1(OE) sensor constructs by replacing GFP in the GFP sensor constructs  sensor construct into mir-71(OE) animals (tir-1(OE) 2AWCOFF phenotype was determined by dividing the 2AWCOFF percentage of tir-1(OE) animals with the 2AWCOFF percentage of their tir-1(OE); mir-71(OE) siblings, which was then normalized to the relative tir-1(OE) transgene copy number determined by qPCR. The higher normalized fold change in tir-1(OE) 2AWCOFF indicates more suppression of 2AWCOFF phenotype by mir-71(OE) in tir-1(OE); mir-71(OE) animals. The normalized fold change in tir-1(OE) 2AWCOFF of tir-1 3′ UTR was significantly higher than that of the tir-1 3′ UTRmut , and 20% had visible YFP in only AWC right (AWCR)  . These rmir-71, when detected in both AWC neurons, has differential expression levels between AWCON and AWCOFF. Transgenic animals expressing mir-71p::GFP, ceh-36p::myr-TagRFP (myristoylated TagRFP marker of AWCON and AWCOFF), and str-2p::2Xnls-TagRFP  were generated and analyzed in the L1 stage  mir-71p::GFP result, recGFP expression was significantly higher in the AWCON cell than in the AWCOFF cell in 81% of the animals , odr-3p::2Xnls-TagRFP::unc-54 3′ UTR , and str-2p::myr-mCherry (myristoylated mCherry marker of AWCON) were generated and analyzed in the L1 stage  construct odr-3p::mir-71 and a mosaic marker odr-1p::DsRed that showed which AWC cells retained the transgene. We specifically looked for the mosaic animals in which only one of the two AWC neurons expressed the mir-71(OE) transgene; this cell was identified by expression of the DsRed marker.To determine the site of mir-71(OE) transgene in a wild-type background. Expression of the mir-71(OE) transgene in both AWC neurons resulted in a 2AWCON phenotype (mir-71(OE) transgene was retained in only one of the two AWC neurons, the mir-71(OE) AWC neuron became AWCON and wild-type AWC neuron became AWCOFF in the majority of these mosaic animals  transgene in nsy-4(ky627) and nsy-5(ky634) mutants. Loss-of-function mutations in nsy-4 and nsy-5 caused a 2AWCOFF phenotype (mir-71(OE) 2AWCON phenotype. Overexpression of mir-71 in both AWC neurons significantly suppressed the 2AWCOFF phenotype of nsy-4(ky627) and nsy-5(ky634) mutants. In addition, nsy-4(ky627); mir-71(OE) and nsy-5(ky634); mir-71(OE) animals resembled the mir-71(OE) parent more closely than the nsy-4(ky627) or nsy-5(ky634) parent, but mixed phenotypes were observed  transgene in only one of the two AWC neurons, the mir-71(OE) AWC neuron expressed str-2p::GFP and the other AWC neuron did not (mir-71 in the future AWCON neuron in nsy-4(ky627) and nsy-5(ky634) mutants is the same as in the wild-type background. These results suggest that mir-71 acts cell autonomously downstream of nsy-4 and nsy-5 to promote the AWCON identity.NSY-4 claudin-like protein and NSY-5 gap junction protein are the two parallel signaling systems that antagonize the calcium signaling pathway to specify the AWChenotype  [18],  IkyIs323  IIoyIs44 [odr-1p::DsRed] VkyIs136  Xmir-71(n4115) Insy-5(ky634) Ippfr-1(tm2180) unc-29(e1072) I  rol-6(e187) II, tir-1(ky388ts) IIItir-1(ky648gf) III, tir-1(tm3036) IIIunc-36(e251) III, dcr-1(ok247) III; nsy-4(ky616) IV, nsy-4(ky627) IVunc-43(n498gf) IV, eri-1(mg366 IV), unc-76(e911) V, lin-15b(n744) X, and alg-1(gk214) X.Wild-type strains were kyEx1127  vyEx149 , vyEx187 , vyEx527, 528 , vyEx605, 606 , vyEx611, 615 , vyEx647 , vyEx649, 651 , vyEx835, 836, 838 , vyEx703, 720 , vyEx905, 907 , vyEx914, 917 , vyEx915, 918 , vyEx916, 920, 921 , vyEx922, 923, 924 , vyEx927, 931 , vyEx1316, 1317 , vyEx1065 , vyEx1097 , vyEx1351, 1352 , and vyEx1353, 1375 .Transgenes maintained as extrachromosomal arrays include mir-71 promoter was subcloned to make mir-71p::YFP and mir-71p::GFP. mir-71p::NZGFP was made by replacing GFP in mir-71p::GFP with a NZGFP fragment from TU#710 (Addgene) odr-3p::CZGFP was made by cloning a CZGFP fragment from TU#711 (Addgene) odr-3p vector. ceh-36p::myr-TagRFP, in which the 1852 bp ceh-36 promoter drives expression of myristoylated TagRFP, was generated by replacing TagRFP in ceh-36p::TagRFPmyr-TagRFP. odr-3p::2Xnls-TagRFP was made by replacing the str-2 promoter in str-2p::2Xnls-TagRFPodr-3 promoter str-2p::myr-mCherry was generated by replacing GFP in str-2p::GFPmyr-mCherry fragment. A 94 bp mir-71 PCR fragment was subcloned to make odr-3p::mir-71. A 561 bp PCR fragment of the tir-1 3′ UTR, which represents the average length of the 3′ UTR in the majority of identified tir-1 cDNA clones such as yk1473h08 (www.wormbase.org), was subcloned to make odr-3p::tir-1::tir-1 3′ UTR and odr-3p::GFP::tir-1 3′ UTR. miRNA target prediction algorithms including MicroCosm Targets, PicTar, and mirWIP use 300–590 bp of tir-1 3′ UTR for analysis. The predicted mir-71 binding site, TCTTTC, in the tir-1 3′ UTR was mutated into CAGGCA using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) to make odr-3p::GFP::tir-1 3′ UTRmut. tir-1a splice form was used for all tir-1 constructs. odr-3p::tir-1 5′ UTR::GFP::tir-1 3′ UTR was made by cloning a 150 bp PCR fragment of tir-1 5′ UTR, amplified from wild-type embryo cDNA, into the odr-3p::GFP::tir-1 3′ UTR. odr-3p::tir-1 5′ UTR::GFP::tir-1 3′ UTRmut was made by replacing GFP::tir-1 3′ UTR in the odr-3p::tir-1 5′ UTR::GFP::tir-1 3′ UTR with GFP::tir-1 3′ UTRmut. To make shRNA anti-mir-248 (mir-248IR), the sense and antisense oligos, each consisting of mir-248 sense (24 nt) and antisense (24 nt) sequences that flank a 12 nt linker (loop) sequence, were designed (SBI System Biosciences) and annealed (IDT) as described. This hairpin construct was subcloned to make nsy-5p::mir-248IR. To generate transgenic strains, DNA constructs were injected into the syncytial gonad of adult worms as previously described A 2476 bp PCR fragment of Z-stack images of transgenic animals expressing fluorescent markers were acquired using a Zeiss Axio Imager Z1 microscope equipped with a motorized focus drive and a Zeiss AxioCam MRm CCD digital camera. All animals of each set of experiments had the same exposure time for comparison of fluorescence intensity. The single focal plane with the brightest fluorescence in each AWC cell was selected from the acquired image stack and measured for fluorescence intensity. To measure fluorescence intensity, the outline spline tool in the Zeiss AxioVision Rel 4.7 image analysis software was used to draw around the AWC cell body  or nucleodr-3p::mir-71; odr-1p::DsRed transgene were passed for minimum of six generations before scoring for mosaic animals. The same transgenic lines were crossed into nsy-4(ky627) and nsy-5(ky634) mutants for the analysis.Mosaic analysis was performed as previously described tir-1(OE) transgene line were collected in 25 µl of worm lysis buffer . Collected worms were then incubated at −80°C for minimum of one hour, 65°C for one hour, and 95°C for 15 minutes. 5 µl of the worm lysate was used for subsequent qPCR with Fast SYBR Green Master Mix (Invitrogen). qPCR reactions were run in triplicate at 95°C for 3 minutes, followed by 45 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds on the CFX96 Real-Time PCR Detection System (Bio-Rad). PCR product was scanned for fluorescent signal at the end of each cycle and the C(T) values were obtained using the CFX Manager Software (Bio-Rad). The relative tir-1(OE) transgene copy number was determined using the 2[−Delta Delta C(T)] method as previously described arx-1, as internal control.Three adult hermaphrodites from each mir-71. The odr-3p::mir-71 transgenes used in genetic mosaic analysis were crossed into various genetic backgrounds. Total RNA samples were isolated from first stage larvae using RNeasy Mini kit (QIAGEN). Reverse transcription (RT) reactions were performed with 1 µg of total RNA, SuperScript III reverse transcriptase (Invitrogen), and RT primer (oligo d(T)18, premature mir-71 stem-loop RT primer, or mature mir-71 stem-loop RT primer). 1 µl of 1∶35 diluted reverse transcription product was used as template for subsequent qPCR reactions with Fast SYBR Green Master Mix (Invitrogen). All PCR reactions were run in triplicate at 95°C for 3 minutes, followed by 45 cycles of 95°C for 30 seconds, 51°C for 30 seconds, and 72°C for 30 seconds on the CFX96 Real-Time PCR Detection System (Bio-Rad). PCR product was scanned for fluorescent signal at the end of each cycle and the C(T) values were obtained using the CFX Manager Software (Bio-Rad). The actin-related gene, arx-1, was used as internal control to normalize variation between samples. Relative expression of premature and mature mir-71 was analyzed using the 2[−Delta Delta C(T)] method as previously described mir-71(n4115); odr3p::mir-71 and was normalized accordingly for other samples. Student's t-test was used to calculate statistical significance.Stem-loop RT-qPCR was performed as described Figure S1miRNAs predicted to target genes in the AWC calcium-mediated signaling pathway. (A) A list of miRNAs and target genes identified by four miRNA target prediction programs. Only the prediction that fits the two indicated criteria is listed. (B) AWC phenotypes caused by overexpression of candidate miRNAs listed in (A).(TIF)Click here for additional data file.Figure S2mir-71 on GFP sensor constructs with the tir-1 3′ UTR or the nsy-1 3′ UTR. (A) Normalized GFP intensity in wild type and mir-71(n4115) mutants carrying the transgene of GFP sensor constructs with the tir-1 3′ UTR or the unc-54 3′ UTR (as negative control). (B) Normalized GFP intensity in wild type and mir-71(OE) animals expressing the transgene of a GFP sensor construct with the nsy-1 3′ UTR.The effect of (TIF)Click here for additional data file.Figure S3tir-1 5′ UTR does not affect mir-71(OE)-mediated downregulation of gene expression through the tir-1 3′ UTR.  The average normalized GFP intensity in the AWC cell body of sensor constructs, driven by the odr-3 promoter and the tir-1 5′ UTR, with the tir-1 3′ UTR (A) or the tir-1 3′ UTR mutated in the predicted mir-71 target site (B), in wild type and mir-71(OE) animals. The GFP intensity of an individual cell was normalized to the TagRFP intensity of the internal control transgene odr-3p::2Xnls-TagRFP::unc-54 3′ UTR in the same cell in the first larval stage. For each sensor construct, the normalized GFP intensity in wild type was set as 1 arbitrary unit (AU) and the normalized GFP intensity in mir-71(OE) was calibrated to that in wild type. Two independent lines were analyzed for each sensor construct. Student's t-test was used for statistical analysis. Error bars, standard error of the mean. ns, not significant.The (TIF)Click here for additional data file.Figure S4mir-71 is higher in the AWCON cell than in the AWCOFF cell.  Images of recGFP expressed from mir-71p::NZGFP and odr-3p::CZGFP.  Images of str-2p::2Xnls-TagRFP. AWCON was identified as str-2p::2Xnls-TagRFP positive (A′). AWCOFF was identified as str-2p::2Xnls-TagRFP negative (B′). (A″) Merge of A and A′ images from the same cell. (B″) Merge of B and B′ images from the same cell. (C) Quantification of recGFP expression in AWCON and AWCOFF cells. All images were taken from first stage larvae. The single focal plane with the brightest fluorescence in each AWC was selected from the acquired image stack and measured for fluorescence intensity. Each animal was categorized into one of three categories: AWCON = AWCOFF, AWCON>AWCOFF, and AWCOFF>AWCON based on the comparison of recGFP intensities between AWCON and AWCOFF cells of the same animal. We did not observe any animals that fell into the “AWCON = AWCOFF” category from our recGFP intensity analysis. Total number of animals for each category was tabulated and analyzed as described p-values were calculated using X2 test. Error bars represent standard error of proportion. Scale bar, 2 µm.The expression level of (TIF)Click here for additional data file.Figure S5mir-71 levels.  Representative images of stem-loop RT-PCR product of total RNA samples from adult worms (A) or enriched first stage larvae (B) in different genetic backgrounds. + and − indicate the presence and absence of the transgene odr-3p::mir-71, respectively. The actin-related gene arx-1 was used as internal control to normalize the abundance of mature mir-71. All PCR reactions were run in triplicate. p values were calculated using Student's t-test. ns, not significant. Error bars represent standard error of the mean.Stem-loop RT-PCR analysis of mature (TIF)Click here for additional data file.Figure S6mir-71 in nsy-4(ky627) and nsy-5(ky634) mutants is not caused by a reduced transmission rate of the odr-3p::mir-71 extrachromosomal array or reduced activity of the odr-3 promoter. (A) Transmission rates of the odr-3p::mir-71 extrachromosomal array in mir-71(n4115), mir-71(n4115);nsy-4(ky627), and mir-71(n4115);nsy-5(ky634) mutants. Error bars represent the standard error of proportion. (B) Top: Representative images of odr-3p::GFP expression in AWC neurons of wild type, nsy-4(ky627), and nsy-5(ky634) mutants at the first larval stage. Bottom: The average intensity of GFP in AWC neurons. Results from two independent odr-3p::GFP transgenic lines are shown. Error bars represent standard error of the mean. Scale bar, 10 µm.Control experiments to demonstrate that a decreased level of mature (TIF)Click here for additional data file.Figure S7mir-71 in the two AWC cells is not dependent on nsy-4 or nsy-5. The GFP intensity of mir-71p::GFP was compared between the two AWC cells of the same animal in wild-type, nsy-4(ky627), and nsy-5(ky634) mutants. The percentage difference of mir-71p::GFP expression between the two AWC cells was determined by dividing the higher GFP intensity with the lower GFP intensity. Error bars represent the standard error of proportion.Differential expression of (TIF)Click here for additional data file.Text S1mir-71 by stem-loop RT–PCR.Supplemental Methods: Quantification of mature (DOCX)Click here for additional data file.