Our findings warrant additional studies to determine if K8/K18 could be a predictor of tumor resistance to DR5-targeted therapies.TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis by engaging its death receptors (DRs) 4 and/or 5 on targeted cells. Clinical attempts to stimulate this apoptotic pathway for cancer therapy, including the use of recombinant human TRAIL (rhTRAIL) or receptor agonistic antibodies, have been underway for over a decade. Unfortunately, these agents have only shown limited therapeutic effects due largely to tumor resistance arising from mechanisms yet to be defined. Here we show that intermediate filament proteins, keratin 8 and keratin 18 (K8/K18), negatively regulate TRAIL induced apoptosis. K8/K18 protein levels are consistently higher in TRAIL-resistant cells compared to TRAIL-sensitive cells in a panel of breast cancer cell lines. Blockade of K8 increased expression of DR5 on the surface of targeted cells and sensitized the cells to TRAIL-induced apoptosis. Conversely, ectopic expression of K8/K18 downregulated DR5 protein expression. K8/K18 appears to negatively regulate apoptosis signaling  Death receptors (DRs) 4 and 5 are cell surface receptors that transmit apoptotic signals initiated by their cognate ligand TNF-related apoptosis inducing ligand (TRAIL). At the molecular level, TRAIL ligation induces DR4/DR5 clustering and subsequently recruits Fas-associated Death Domain protein (FADD) and caspase-8 forming the death-inducing signaling complex (DISC). Proximity-induced autoactivation of caspase-8 within the DISC triggers activation of executioner caspases-3 and -7, which cleave structural proteins ultimately leading to apoptotic cell death . A body In this study, we identified a novel role of keratins in regulating TRAIL induced apoptosis. Keratins are the major intermediate filament (IF) proteins of the cytoskeleton, where keratin 8 and 18 (K8/K18) pair is preferentially expressed in simple epithelia cells of gastrointestinal tract, liver, pancreas, and mammary gland, from which many carcinomas arise -9. In thThe intermediate filament protein pair keratin 8 and 18 (K8/K18) have been linked to cellular resistance to several chemotherapeutic drugs -12 and tKRT8) into K8/K18-expressing breast cancer cell lines . Immunoblot analysis confirmed effective K8 knockdown in targeted cell lines  against keratin 8 gene  as a functional regulator of apoptosis via DR5  but are also active regulators of cancer cell signaling -9. The l5 Figure  & 4. ConAs a death receptor, DR5 must be expressed on the surface membrane to interact with incoming death ligand . However, several reports revealed DR5 localization in nuclei and other intracellular compartments -29. ImpoThe cytoskeletal network can interact and regulate death receptors through multiple mechanisms Figure . Our labThe human breast cancer cell lines including AU565, BT474, MDA-MB-453, HCC1428, MDA-MB-361, T47D, MCF7, ZR751, HCC1500, HCC1937, HCC1954, MDA-MB-468, SKBR3, MDA-MB-157, MDA-MB-231, HCC38, HCC2157, and BT549 were purchased from American Type Culture Collection (ATCC). All cell lines were cultured per ATCC recommendation and were tested for the absence of mycoplasma contamination regularly. Recombinant human TRAIL protein, containing amino acids 114-281 of human TRAIL was produced by E. coli as homotrimers and was purchased from R&D Systems (375-TEC). Antibodies specific to human caspase-3 (8G10), caspase-8 (1C12), DR4 (D9S1R), and DR5 (D4E9) were purchased from Cell Signaling Technology. Keratin 8/18 antibodies were purchased from Cell Signaling (C51) and BioLegend (1E8). GAPDH antibody was purchased from Novus (2D4A7). Horseradish peroxidase–conjugated goat anti-rabbit IgG1 (sc-2054), goat anti-mouse IgG1 (sc-2969), and donkey anti-goat IgG (sc-2020) were purchased from Santa Cruz Biotechnology. Phycoerythrin (PE)-conjugated monoclonal antibodies to DR4 (FAB347P) and DR5 (FAB6311P) and corresponding IgG1 (IC002P) and IgG2b (IC0041P) controls were purchased from R&D Systems.2 Profiler Apoptosis PCR array through contract service with Qiagen.mRNA expression was determined using a RTSynthetic small interference RNA (siRNA) oligos specific to keratin 8 (GAAGCAACAUGGACAACAUTT) or validated nontargeting control siRNA was purchased from ThermoFisher Scientific. Cells were grown to 50-70% confluence and transfected with siRNA oligos for 72 hours using lipofectamine. When indicated, cells were incubated with rhTRAIL (100 or 150 ng/ml) for an additional 24 h. Cells were harvested using non-enzymatic cell dissociation solution, Cellstripper (Corning).pCMV6-ENTRY cDNA plasmid encoding human keratin 18 and a control plasmid were purchased from Origene Technologies. MDA-MB-231 cells were grown in 6-well plates to 70 % confluence and were transfected with plasmids using X-tremeGENE HP DNA transfection reagent (Millipore Sigma) for a duration of 24 h. As needed, transfected cells were incubated with rhTRAIL  for 3 hours followed by immunoblotting for caspase activation.t-test.Cells were lysed using RIPA buffer  (Millipore). Cellular debris was removed by centrifugation and total protein amount was determined by BCA assay (ThermoFisher Scientific). Equal amount of total protein (20-30 μg) was resolved by SDS-PAGE using NuPAGE 4–12% gradient Bis-Tris gels (Life Technologies) and transferred to PVDF membranes. Membranes were blotted with appropriate antibody and visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and an ImageQuant LAS 4000 imager (GE). Protein expression levels were quantified by densitometry analysis of immunoblots using the imager built-in software and were normalized to the corresponding actin or GAPDH loading controls. P-values were calculated using student’s Cell pellets were lysed in RIPA buffer. In 1 ml total volume, 1 mg of protein lysate was incubated with 6 μg of anti-K8/K18 antibody, purchased from BioLegend (1E8) for 4 h at 4 °C. 60 μl of protein A-agarose slurry (Santa Cruz) was then added and further incubated overnight at 4 °C. The beads were separated from the lysate using centrifugation and were washed 5 times using lysis buffer. After the last wash beads were treated with 1 X LDS sample buffer and heated at 90 °C for 10 minutes. IP samples were analyzed by SDS-PAGE and immunoblot analysis as previously described.To quantify apoptosis, cells were labeled with Annexin V-FITC and propidium iodide (PI) using an eBioscience Apoptosis kit (88-8007-72). Labeled cells were analyzed using BD Accuri C6 flow cytometer (BD). Flow cytometry analysis of surface receptors was performed as previously described. Briefly, cells were harvested using non-enzymatic cell dissociation solution Cellstripper (Corning). Cell pellets were blocked in PBS buffer containing 5% normal goat serum and 1 % bovine serum albumin on ice for 20 minutes. Cells were then incubated on ice for 45 minutes in the dark with phycoerythrin (PE)-conjugated anti-DR4 or anti-DR5 monoclonal antibodies or corresponding PE-conjugated isotypes. Cells were subsequently washed twice with PBS buffer, resuspended in PBS buffer containing 1 % bovine serum albumin and analyzed.Cells were seeded onto sterilized coverslips coated with poly-L-lysine. Attached cells were fixed with 4 % paraformaldehyde in PBS buffer for 15 minutes and were permeabilized using PBS buffer containing 0.5 % triton X-100 for 15 minutes. Cells were then blocked with PBS buffer containing 3 % BSA and were labeled with specific primary antibodies, anti-keratin 8 rabbit mAb  and anti-DR5 mouse mAb . Alexa Fluor 594 goat anti-mouse and Alexa 488 goat anti-rabbit antibodies were used as secondary antibodies for detection (ThermoFisher Scientific). Nuclei were stained with DAPI and actin was stained with phalloidin conjugated to Alexa 488 dye. Confocal images were captured using an LSM 510 Meta Confocal Microscope attached to an Axiovert 200 Inverted Microscope (Carl Zeiss). An oil immersion 40x objective lens was used for all optical imaging. Laser lines of 405, 488, and 594 were used to detect DAPI, Alexa 488, and Alexa 594 respectively.


Scientific Reports 10.1038/s41598-017-19137-5, published online 12 January 2017Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:“This work has been supported by the Bavarian Research Foundation (AZ-1200-15)”.


LacZ/LacZ) or wild-type mice. These mice were euthanized 16 weeks after inducing hyperglycemia. Increased urine albumin excretion and creatinine clearance observed in diabetic wild-type mice were significantly prevented in diabetic VASH2-deficient mice. Accordingly, diabetes-induced increase in glomerular volume and reduction in glomerular slit-diaphragm density were significantly improved in VASH2 knockout mice. Increased glomerular endothelial area was also suppressed in VASH2-deficient mice, in association with inhibition of enhanced vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), but not VEGF level. Furthermore, glomerular accumulation of mesangial matrix, including type IV collagen, and increased expression of TGF-β were improved in diabetic VASH2 knockout mice compared with diabetic wild-type mice. Based on the immunofluorescence findings, endogenous VASH2 localization in glomeruli was consistent with mesangial cells. Human mesangial cells (HMCs) were cultured under high glucose condition in in vitro experiments. Transfection of VASH2 small interfering RNA (siRNA) into the HMCs resulted in the suppression of type IV collagen production induced by high glucose compared with control siRNA. These results indicate that VASH2 may be involved in diabetes-induced glomerular alterations, particularly impaired filtration barrier and mesangial expansion. Therefore, VASH2 is likely to represent a promising therapeutic target for diabetic nephropathy.Angiogenesis has been implicated in glomerular alterations in the early stage of diabetic nephropathy. We previously reported the renoprotective effects of vasohibin-1 (VASH1), which is a novel angiogenesis inhibitor derived from endothelial cells, on diabetic nephropathy progression. Vasohibin-2 (VASH2) was originally identified as a VASH1 homolog and possesses pro-angiogenic activity in contrast to VASH1. In addition, VASH2 was recently shown to promote epithelial-to-mesenchymal transition via enhanced transforming growth factor (TGF)-β signaling in cancer cells. Herein, we investigated the pathogenic roles of VASH2 in diabetic nephropathy using VAHS2-deficient mice. The type 1 diabetes model was induced by intraperitoneal injections of streptozotocin in VASH2 homozygous knockout (VASH2 Diabetic nephropathy is a leading cause of end-stage kidney disease (ESKD) in developed countries. In earlier stage, glomerular hyperfiltration, glomerular hypertrophy, glomerular basement membrane (GBM) thickening, and microalbuminuria are generally observed, followed by mesangial matrix expansion and proteinuria. Subsequently, nodular glomerulosclerosis and massive proteinuria develop in the advanced stage, leading to ESKD . DespiteAngiogenesis, the growth of new blood vessels from pre-existing vessels, is associated with a number of pathological processes, and is also involved in the pathogenesis of diabetic nephropathy. Previous studies demonstrated new capillary formation and pre-existing capillary elongation , 4, as wVasohibin-1 (VASH1) is a unique endothelium-derived angiogenesis inhibitor, which prevents proliferation and migration of endothelial cells in an autocrine manner , 11. We In the present study, we have demonstrated the improvement of diabetic nephropathy in VASH2-deficient mice, and the inhibition of high glucose-induced extracellular matrix (ECM) protein production in cultured mesangial cells with suppressed VASH2 expression.LacZ/LacZ) mice were obtained from the Institute of Development, Aging, and Cancer, Tohoku University . The experimental protocol was approved by the Animal Care and Use Committee in Okayama University . Male C57BL/6J wild-type (WT) and C57BL/6J VASH-2LacZ/LacZ mice were fed a standard pellet laboratory chow and were provided with water ad libitum. Type 1 diabetes was induced by intraperitoneal injection of 50 mg/kg streptozotocin  for five consecutive days [LacZ/LacZ (V2KO-NDM), (3) diabetic WT (WT-DM) and (4) diabetic VASH-2LacZ/LacZ (V2KO-DM) mice. In VASH-2 homozygous knockout . Serum and urine creatinine levels, urine albumin concentration, and HbA1c were determined as previously described . CreatinGV) was determined from the mean glomerular cross-sectional tuft area (GA), as previously described [GA. GV was calculated by the following equation:k = 1.1 is a size distribution coefficient. GV was expressed relative to the value of the WT-NDM group.10% buffered formalin-fixed and paraffin-embedded 4 μm sections were stained with periodic acid Schiff (PAS) for light microscopy observation. The mean glomerular volume , as previously described . The folTransmission electron microscopy was performed as previously described . The kidImmunoblot assay was performed, as previously described . (1) ant5ʼ-GTG TGG AGC AAC ATG TGG AAC TCT A-3ʼ (forward) and 5’-TTG GTT CAG CCA CTG CCG TA-3ʼ (reverse); mouse VASH2, 5’-GGC TAA GCC TTC AAT TCC CC-3’ (forward) and 5’-CCC ATT GGT GAG ATA GAT GCC-3’ (reverse); mouse 18S rRNA, 5’-ACT CAA CAC GGG AAA CCT CA-3’ (forward) and 5’-AAC CAG ACA AAT CGC TCC AC-3’ (reverse); human VASH2, 5’-ACG TCT CAA AGA TGC TGA GG-3’ (forward) and 5’-TTC TCA CTT GGG TCG GAG AG-3’ (reverse); human type IV collagen α3, 5’-CAG GAC TCA CGG GTT CCA AAG-3’ (forward) and 5’-TGC ATC CTG GTA CAC CGA CAA-3’ (reverse); human connective tissue growth factor (CTGF), 5’- CTT GCG AAG CTG ACC TGG AA-3’ (forward) and 5’- AAA GCT CAA ACT TGA TAG GCT TGG A-3’ (reverse); human 18S rRNA, 5’-ACT CAA CAC GGG AAA CCT CA-3’ (forward) and 5’-AAC CAG ACA AAT CGC TCC AC-3’ (reverse). Real-time PCR amplifications were performed using SYBR Green PCR Master Mix , the abovementioned primers and StepOnePlus™ Real-Time PCR System (Applied Biosystems).RNA extraction and real-time polymerase chain reaction (PCR) were performed, as previously described , 13, wit5’-CAC UCU GAA UGA AGU GGG CUA UCA A-3’, and control siRNA was 5’-CGA CCU GCC CAA GAU UCC CAU ACC A-3’.Human mesangial cells  were cultured in Dulbecco's Modified Eagle's medium  supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml). For the experimental study, human mesangial cells were incubated in DMEM with normal glucose  and high glucose  for 24 hours after serum starvation. Normal glucose plus 19.5mM mannitol (MN) was used as osmotic control. For VASH2 gene-silencing study, cells were transfected with siRNA using Stealth Select RNAi , as previously described . The nucP <0.05 was considered statistically significant.All values are expressed as the mean ± standard error (SE). A Kruskal Wallis test with post-hoc comparisons using the Scheffe’s test was utilized for inter-group comparisons of multiple variables. The statistical analysis was performed using the JMP 10 software . A level of STZ-induced diabetes resulted in significantly increased hemoglobin A1c (HbA1c) and, systolic blood pressure (SBP), and body weight (BW) loss, but did not change the serum creatinine (sCr) level. VASH2-deficiency did not affect BW, SBP, HbA1c, and sCr compared with WT mice. When diabetes was induced, no differences in these parameters were found between diabetic WT and VASH2-deficient mice . In non-No differences in glomerular histology between non-diabetic WT and VASH2-deficient mice were observed on light microscopy . DiabeteConsidering previous reports that VASH2 directly acts on endothelial cells to exert proangiogenic properties , alteratVEGF-A is essential for the integrity of glomerular capillary structure and function . In diabConsistent with glomerular mesangial expansion on light microscopy, diabetes induced prominent glomerular deposition of type IV collagen in WT mice . IncreasEndogenous VASH2 mRNA expression in renal cortex was significantly up-regulated in diabetic mice . BecauseIn vitro experiments using cultured human mesangial cells (HMCs) under normal glucose (NG) or high glucose (HG) condition were performed to clarify the potential role of VASH2 expressed in mesangial cells. The 24-hour incubation in HG condition significantly increased VASH2 mRNA expression in cultured HMCs compared with NG condition. However, addition of mannitol (MN) to NG condition could not induce the upregulation of VASH2 expression , with 52.5% of the homology between the two protein sequences . In contOne of the most notable findings in this study was the prevention of diabetes-induced urine albumin excretion in VASH2-deficient mice. A unique crosstalk between endothelial cells and podocytes has been recognized to maintain integrity of glomerular filtration barrier , 28. In in vitro experiment demonstrated that knockdown of VASH2 in mesangial cells prevented the increased mRNA level of type IV collagen induced by high glucose concentration. VASH2 has been reported to increase the expression of TGF-β type I receptor and promote the downstream signaling [Mesangial expansion is a characteristic glomerular alteration in diabetic nephropathy, leading to glomerulosclerosis . Mesangiignaling . These rAs described above, no specific phenotype in non-diabetic condition was observed in VASH2-deficient mice compared with WT mice, consistent with other previous reports , 17. TheThis study has several limitations. First, we evaluated the role of endogenous VASH2 in a type 1 diabetes model, and thus, the use of type 2 diabetes model should be considered in the future. Second, as we utilized the traditional  gene knockout mice in this study, whether our results might be ascribed to circulating VASH2 or renal VASH2 remains unclear. Considering up-regulation of VASH2 in cultured mesangial cells treated with high glucose concentration, renal VASH2 should play significant roles in diabetic nephropathy. However, further experiments using conditionally knocked out VASH2 mice are warranted.In conclusion, endogenous VASH2 may exacerbate urine albumin excretion and mesangial expansion in diabetic nephropathy, possibly through enhancing VEGFR-2 signaling in glomerular endothelial cells and ECM production in mesangial cells, thus indicating that VASH2 could represent a potential therapeutic target for diabetic nephropathy.


PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN) and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23). Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.Protocadherin 15 (PCDH15) is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of Inner ear hair cells are responsible for mechanoelectrical transduction (MET) that converts mechanical stimuli into electrical signals. MET occurs within stereocilia, the F-actin-based, and microvilli-like protrusions on the apical surface of hair cells . Stereoc PCDH15 gene are responsible for syndromic hearing loss Usher 1F or nonsyndromic hearing loss DFNB23 [ Pcdh15 mutations are also responsible for the hearing and balancing deficits of Ames waltzer (av) mice [Protocadherin 15 (PCDH15) is an atypical cadherin that contains eleven extracellular cadherin (EC) repeats, whereas classical cadherins usually have only five EC repeats. Genetic, biochemical, immunochemical, and structural evidence indicated that PCDH15 and cadherin 23 (CDH23), another atypical cadherin, form the lower and higher part of tip-links, respectively . PCDH15 s DFNB23 –12. Pcdhav) mice , 14. In Three prominent PCDH15 isoforms are generated by alternative pre-mRNA splicing, which are PCDH15-CD1, PCDH15-CD2, and PCDH15-CD3 . These P harmonin knockout mice [ myosin VIIA mutant mice [Several other PCDH15-binding proteins have also been identified so far. PCDH15-CD1 was shown to interact with PDZ domain-containing protein harmonin via its C-terminal PBI , 23. Furout mice . PCDH15-ant mice . Recentlant mice . In the  Pcdh15-CD3 cDNA was inserted into pBD-GAL4 Cam vector (Stratagene) to express the C-terminal 106 amino acids of chicken PCDH15-CD3 as bait protein. Mouse Pcdh15 cDNAs were inserted into pcDNA3.1(+) to express full length PCDH15 isoforms with a Myc tag between the N-terminal signal peptide and the first EC repeat. Mouse Pcdh15 cDNAs were inserted into pEGFP-C2 or modified pEGFP-C2 (EGFP replaced with Myc) to express EGFP-tagged or Myc-tagged PCDH15 cytoplasmic domains. The cDNA encoding mouse PCDH15-CD3 missing the last 4 aa (MTKL) at the C-terminus was inserted into the same vectors to express Myc-PCDH15-CD3 (-MTKL) and Myc-PCDH15-CD3 Cter (-MTKL). Human PIST cDNA was inserted into pEGFP-C2 or modified pEGFP-C2 to express full length PIST, PIST CC2-plus domain (146-274aa), and PIST PDZ domain (276-366aa) as EGFP-fusion or Myc-fusion proteins. Mouse monoclonal anti-EGFP antibody (Cat. number M20004) was from Abmart. Mouse monoclonal anti-Myc antibody (Cat. number M4439) was from Sigma-Aldrich.Chicken6 transformants were selectively screened using HIS3  as the primary reporter gene. The positive colonies were further examined using two more reporter genes ADE2 and lacZ. The prey vectors in triple-positive yeast colonies were recovered and the sequence of cDNA inserts was determined by Sanger sequencing.The yeast two-hybrid screen was performed as previously described , 28. BriHEK293T cells were transfected with the expression vectors using jetPRIME Transfection Agent  according to the manufacturer's instructions. Transfected cells were washed with phosphate-buffered saline (PBS) 24 hours after transfection and lysed in ice-cold lysis buffer consisting of 150 mM NaCl, 50 mM Tris at pH 7.5, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1 × protease inhibitor cocktail (Roche). After centrifugation at 4°C, the supernatant was collected and incubated with immobilized anti-Myc antibody  at 4°C overnight. After washing five times with washing buffer (a modified lysis buffer containing 500 mM NaCl instead of 150 mM), the immunoprecipitated proteins were separated by polyacrylamide gel electrophoresis (PAGE) and then transferred to PVDF membrane. The blot was incubated with corresponding primary antibodies, followed by incubation with secondary antibodies (Bio-Rad), and the signals were detected with the ECL system .COS-7 cells were grown on Gelatin-coated glass cover slips and transfected with the expression vectors. Transfected cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 minutes and then permeabilized and blocked with PBT1  for 30 minutes, followed by incubation with primary antibody diluted in PBT1 over night at 4°C. After washing twice with PBT1 for 10 minutes and twice with PBT2  for 5 minutes, cells were incubated with fluorescence-conjugated secondary antibody (Life Tech) in PBT2 for 1 hour, followed by two 5-minute PBT2 washes and two 5-minute PBS washes. For nuclei staining, cells were incubated with DAPI (Gen-View Scientific Inc.) for 15 minutes, followed by four 5-minute PBS washes and then mounted in Glycerol/PBS (1 : 1). The cells were imaged with a confocal microscope . HIS3 gene was used as the primary reporter gene for the screen in the presence of 3-amino-1,2,4-triazole (3-AT) that inhibits the autoactivation of HIS3 reporter gene. When the intact cytoplasmic domain of chicken PCDH15-CD3 was used as bait, it autoactivated the HIS3 reporter gene at the highest 3-AT concentration (15 mM) tested. Then various regions within the PCDH15-CD3 cytoplasmic domain were tested for autoactivation. The results showed that the fragment containing the C-terminal 106 amino acids does not activate HIS3 reporter gene at the presence of 2.5 mM 3-AT, and this fragment was used in the following yeast two-hybrid screen. Among the positive clones that activate all the three reporter genes , several clones encode the PDZ-containing, Golgi-associated chaperone protein PIST. PIST has been shown to play important roles in regulating the membrane targeting of several transmembrane proteins such as frizzled, somatostatin receptor subtype 5 (SSTR5) and β1-adrenergic receptor (β1AR) [To identify proteins that interact with PCDH15, we performed yeast two-hybrid screens of a chicken cochlear cDNA library using PCDH15 as bait.r (β1AR) –32.We then performed coimmunoprecipitation (co-IP) experiments to verify the interaction between PCDH15 and PIST. From here on in the rest of our investigation, we focused on mammalian proteins. Our data showed that when overexpressed in HEK293T cells, EGFP-tagged cytoplasmic domain of mouse PCDH15-CD3 was co-IPed with Myc-tagged human PIST, whereas PCDH15-CD1 and PCDH15-CD2 were not Figures . This rePreviously we have shown that PIST binds to cadherin 23 (CDH23), the upper tip-link component, and retains CDH23 in the trans-Golgi network (TGN) in cultured cells . To testWe then performed co-IP experiments to explore which regions/motifs in PCDH15-CD3 and PIST are responsible for the interaction between them. PIST contains a PDZ domain near the C-terminus and two coiled-coil (CC) domains in the middle part . Co-IP eFinally, we examined whether the association with PIST affects the membrane targeting of PCDH15-CD3. When coexpressed with EGFP, Myc-PCDH15-CD3 mainly localizes in the cytoplasm and on the plasma membrane . The plaPCDH15 and CDH23 form the lower and upper part of tip-links, respectively, and are indispensable for hearing transduction. Besides tip-links, PCDH15 and CDH23 also localize at kinociliary-links, transient lateral-links, and ribbon synapse of hair cells , 35–37. In the present work we show that PIST might play an important role in regulating the intracellular traffic and membrane targeting of PCDH15. Previously we have shown that PIST regulates the membrane expression of CDH23 . Taken tInterestingly, we found that, besides the PDZ domain, the CC2 domain and its downstream amino acids (CC2-plus) of PIST also interact with PCDH15-CD3. This was also observed in the interaction between PIST and CDH23 . Neverth


Quantitative real-time PCR analysis of 24 primary EC tissues showed that lower let-7b and miR-361 levels were associated with worse patient outcomes. These results were validated in a larger EC patient dataset from The Cancer Genome Atlas. Our findings suggest that EZH2 drives EC progression by regulating miR-361/Twist signaling, and support EZH2 inhibition as a promising anti-EC therapeutic strategy.EZH2 inhibition and reactivation of tumor suppressor microRNAs (miRNAs) represent attractive anti-cancer therapeutic strategies. We found that EZH2-suppressed let 7b and miR-361, two likely tumor suppressors, inhibited endometrial cancer (EC) cell proliferation and invasion, and abrogated cancer stem cell-like properties. In EC cells, EZH2 induced and functioned together with YY1 to epigenetically suppress miR-361, which upregulated Twist, a direct target of miR-361. Treating EC cells with GSK343, a specific EZH2 inhibitor, mimicked the effects of siRNA-mediated EZH2 knockdown, upregulating miR-361 and downregulating Twist expression. Combining GSK343 with 5 AZA-2′-deoxycytidine synergistically suppressed cell proliferation and invasion  Endometrial cancer (EC) is a heterogeneous disease characterized by dysregulated cell proliferation and metastasis . While aEZH2, and downregulating Twist expression [Enhancer of zeste homolog 2 (EZH2), a crucial component of polycomb repressive complex 2, is highly expressed in multiple cancer types, making it an attractive therapeutic target in tumor treatment . We repopression . EZH2 mapression . EZH2 sipression , suggestin vitro. We report that EZH2 directly downregulates miR-361, a novel Twist suppressor, in EC cells. Our results support the potential clinical use of GSK343 for targeting EZH2 in EC and other human cancers.We identified tumor-suppressive let-7b and miR-361 as EZH2-downregulated miRNAs that attenuate EC cell proliferation, invasiveness, and cancer stem cell-like properties. We further demonstrated that EZH2 upregulates expression of Twist, a direct target of miR-361, via direct repression of miR-361 through a YY1-dependent mechanism. EZH2 inhibition by the specific inhibitor GSK343 is sufficient to induce miR-361 expression, decrease Twist levels and inhibit EC cell proliferation and invasiveness We showed that miR-101 directly targets EZH2 , which cUsing a quantitative real-time PCR (qRT-PCR) assay, we validated that let-7b, miR-361, and miR-378 levels in 24 primary ECs were reduced compared to adjacent normal tissues Figure . We evalin vitro cell invasion and proliferation  mRNAs were upregulated, whereas mesenchymal, stemness, and drug resistance markers  were downregulated in SPAC-1-L cells  and miR-361 mimic were co-transfected into SPAC-1-L cells. miR-361 suppressed Twist 3′-UTR reporter activity , and upregulated IFN-α and IFN-γ, which are known to stimulate antitumor immunity and process anti-angiogenic effects [IL-6 and IL-8 expression, but reduced INF-α and IFN-γ  for YY1 -qPCR assays with antibodies against EZH2, H3K27me3, and YY1 using lysates from SPAC-1-L cells that express endogenous EZH2 and YY1. EZH2, H3K27me3, and YY1 occupied the region of the miR-361 promoter  similar to EZH2/H3K27me3/YY1 binding to the let-7a promoter [miR-361 promoter  upstream of 1 Figure , which rn Figure , suggestpromoter   into a pGL3 luciferase vector , similar to the effects of miR-361 mimics. The opposite effect was detected in Ishikawa cells overexpressing EZH2 -induced hypoacetylation, are linked to gene silencing, we speculated that combinatorial use of epigenetic drugs targeting distinct epigenetic machinery might achieve a synergistic effect on activation of the silenced genes. The HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), stimulates EC cell migration . We confmiR-361, but detected no CpG-enriched region (data not shown), indicating that miR-361 induction by 5-AZA might result from epigenetic activation of an upstream miR-361 regulator. We then investigated the antineoplastic effects of GSK343 (a specific inhibitor of EZH2 methyltransferase activity) [Using a computational bioinformatics analysis, we screened CpG islands upstream of ctivity)  combinedWestern blot analysis demonstrated that GSK343 treatment dose-dependently reduced levels of H3K27Me3, the enzymatic product of EZH2 methyltransferase, without affecting total H3. GSK343 had no effect on EZH2 expression in SPAC-1-L and HOUA-I cells Figure . GSK343 We then surveyed miR-361 and EMT-related marker expression. Similar to EZH2 knockdown, GSK343 treatment enhanced miR-361 and E-cadherin levels, and decreased the expression of Snail, N-cadherin, Twist and Vimentin Figure . EZH2 enin vitro results, we evaluated the effects of GSK343 and 5-AZA alone and combined on EC growth in vivo. Tumors generated by inoculation of Ishikawa cells were treated with the vehicle, GSK343, 5-AZA, or GSK343 plus 5-AZA. As single agents, GSK343 and 5-AZA decreased tumor size and weight compared to vehicle alone, and when combined, increased tumor growth inhibition compared to either agent alone  and those with higher than average miR-361 expression (n = 12). Reduced miR-361 was associated with poorly differentiated histology  and maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS). The EC cell lines, Ishikawa  and HOUA-I , were grown in DMEM/F12  supplemented with 10% FBS. The immortalized human endometrial epithelial cell line, EM, was obtained from Professor Satoru Kyo  and cultured in DMEM/F12 supplemented with 15% FBS. Cells were seeded and incubated for 1 d, then treated with GSK343  and/or 5-AZA  for 3 or 14 d as indicated. Media was changed daily or added every 3 d (sphere formation assay). Transient transfection of miRNA mimic, antisense miRNA inhibitor, and siRNA , or plasmids including pCMV6-AC, pCMV6-TWIST , pCMV/hygro and pCMV/hygro-EZH2 , was accomplished using Lipofectamine 2000 , according to the manufacturer's instructions.http://pga.mgh.harvard.edu/primerbank/).Total RNA was extracted using TRIzol reagent , and was reverse-transcribed into cDNA using the PrimeScript RT reagent kit  according to the manufacturer's instructions. miRNA and mRNA qRT-PCR was performed using NCode miRNA qRT-PCR analysis  and Takara SYBR Premix Ex Taq II , respectively. Forward primers for miRNA detection were exact sequences of mature miRNAs. GAPDH was used for normalization. Primers used for mRNA expression were obtained from the PrimerBank database (3) were plated in 96-well plates for 24 h and then treated with DMSO or varying doses of paclitaxel . After 24 h, cell viability was determined using the Cell Counting Kit-8 . Absorbance was determined at 450 nm using a microplate reader, and percent absorbance was calculated against DMSO-treated cells. Cells were transfected or treated as indicated for 72 h, and cell proliferation was assessed using the Cell Counting Kit-8. In the colony formation assay, approximately 500 cells were added to each well of a 6-well culture plate. Each experiment was performed in triplicate. After 14 d of culture at 37°C, cells were fixed using 10% formalin and then stained using 10% Giemsa. Colonies containing ≥50 cells were counted under a microscope.Cells  for 12 h. Distance migrated was quantified using pictures taken at 0 and 12 h.Cell invasion and migration were monitored and analyzed as described previously , 28. 5 ×Whole-cell or nuclear protein extracts were prepared using the M-Per Mammalian Protein Extraction Reagent  or the Nuclear Extraction Kit  according to the manufacturer's instructions. Total protein (30 μg) and nuclear protein (10 μg) were loaded onto 10–20% SDS-PAGE gels, electrophoresed, and then transferred to nitrocellulose membranes. Antigen-antibody complexes were detected using the enhanced chemiluminescence blotting analysis system . The following antibodies were used: EZH2 , H3K27me3 , total histone 3 , E-cadherin , Vimentin , Twist , p-AKT , AKT , GAPDH , YY1  and lamin B1 . GAPDH (whole cell lysate) and lamin B (nuclear protein) were applied as loading controls. Primary antibodies were used at a dilution of 1:1000.-1) were cultured in serum-free medium supplemented with N2 plus media supplement , epidermal growth factor , basic fibroblast growth factor , and heparin  for 14 d. Spheres > 50 μm were counted.Cells  as previously described [Twist 3′-UTR luciferase vectors were obtained from OriGene (sc209156). To construct pri-miR-361 promoter luciferase reporter vectors, the promoter region encompassing YY1 binding sites  was amplified from human genomic DNA and cloned into pGL3 vector MluI/BglII sites (Promega). A quick-change site-directed mutagenesis kit  was used to mutate the miR-361 binding site within the Twist 3′-UTR or YY1 binding sites on the miR-361 promoter. Luciferase activity was measured 24 h after transfection using the dual-luciferase reporter assay system . The Renilla luciferase reporter plasmid, pRL-CMV, was used to normalize transfection efficiency, and firefly luciferase activity was normalized to Renilla luciferase activity. Primers are provided in GAPDH promoter (negative control). The human let-7a [Chromatin from EC cells was immunoprecipitated with antibodies against EZH2 , H3K27me3 , YY1 (Santa Cruz), or IgG (Santa Cruz) as a control, using the Pierce Agarose ChIP kit  as previously described [n let-7a  promoter6) were suspended in phosphate-buffered saline  and then injected subcutaneously into five-week-old female nude mice (BALB/c). When tumors became palpable, , mice were intraperitoneally injected with control vehicle (DMSO), 5-AZA (0.2 mg kg-1) and/or GSK343 (15 mg kg-1) three times weekly for 2 weeks, after which mice were sacrificed. Tumor length and width were measured using calipers, and tumor volume was calculated using the formula: tumor volume = length × width2 × 0.5. In a parallel experiment, portions of excised tumors embedded in paraffin were subjected to immunohistochemical analysis for Ki-67  expression as previously described [All experiments involving mice were performed in accordance with the guidelines of the animal care and use committee of the Cancer Center, Sun Yat-Sen University. Ishikawa cells 1×10 were susTwenty-four pairs of EC and adjacent non-tumor endometrial tissues were used in this study following review and approval by the Cancer Center, Sun Yat-Sen University. Informed consent was obtained from all patients before operation. Clinical and pathological data are described in t-tests were used for statistical analysis. Fisher exact tests were used to compare categorical data. P < 0.05 represented statistical significance.All experiments were performed in triplicate. Results are expressed as means ± SEMs, and 2-tailed Student's


This transgenic rice line exhibited higher Fe(III) chelate reductase activity and tolerance to Fe deficiency. In addition, we produced transgenic rice overexpressing the Fe deficiency-inducible transcription factor, OsIRO2, which regulates the expression of various genes involved in the strategy II Fe(III) uptake system, including OsNAS1, OsNAAT1, OsDMAS1, OsYSL15, and TOM1. This transgenic rice exhibited improved phytosiderophore secretion ability and tolerance to Fe deficiency. In the present research, transgenic rice plants that possess both the OsIRT1 promoter-refre1/372 and the 35S promoter-OsIRO2 (RI lines) were produced to enhance both Strategy I Fe(II) reductase ability and Strategy II phytosiderophore productivity. RI lines exhibited enhanced tolerance to Fe-deficient conditions at the early and middle-late stages of growth in calcareous soil, compared to both the non-transgenic line and lines harboring either OsIRT1 promoter-refre1/372 or 35S promoter-OsIRO2 alone. RI lines also exhibited a 9-fold higher yield than the non-transgenic line. Moreover, we successfully produced Fe-deficiency-tolerant Tachisugata rice, which is a high-biomass variety used as fodder. Collectively, our results demonstrate that combined enhancement of two Fe uptake systems in rice is highly effective in conferring tolerance to low Fe availability in calcareous soil.Iron (Fe) deficiency is a critical agricultural problem, especially in calcareous soil, which is distributed worldwide. Rice plants take up Fe(II) from soil through a OsIRT1 transporter (Strategy I-related system) and also take up Fe(III) via a phytosiderophore-based system (Strategy II system). However, rice plants are susceptible to low-Fe conditions because they have low Fe(III) reduction activity and low-level phytosiderophore secretion. Previously, we produced transgenic rice plants expressing a mutationally reconstructed yeast ferric chelate reductase,  Iron (Fe) deficiency is a widespread agricultural problem that is common in calcareous soils, which cover more than 30% of the earth’s surface. Although there are abundant minerals in soil, Fe is sparingly soluble under aerobic conditions, in particular at high pH in calcareous soil . Consequ2+  ethanesulfonic acid at pH 5.5, 0.1 mM Fe(III)-EDTA, and 0.2 mM bathophenanthroline disulfonic acid disodium salt) . After incubation for 1 h at 25°C, an aliquot of the assay solution was collected, and the absorbance of the solution at 535 nm was determined using a UV-2450 spectrophotometer . The amount of Fe2+ produced was calculated from a standard curve prepared using standard solutions of Fe2+.Tsukinohikari-RI plants were germinated on MS medium. Two weeks later, plantlets were cultivated in hydroponic culture solution as described above for 10 days under Fe-sufficient conditions, and then cultured in Fe-deficient culture solution without any form of Fe. Root Fe(III)-chelate reductase activities in whole intact root systems were determined as described previously  at 3, 5,2 seeds of Tsukinohikari-RI line Nos. 21 and 22, T2 seeds of Tsukinohikari-Refre1 line No. 7 . n.d., not detected.Non-transgenic line (NT), and transgenic rice expressing the (PDF)Click here for additional data file.S2 Fig35S promoter-OsIRO2 (IRO2), the OsIRT1 promoter-refre1/372’ (Refre1), and the RI lines (21 and 22) were grown in hydroponic culture solution for 10 days and then transferred to hydroponic culture solution without Fe. SPAD values and Fe(III)-chelate reductase activities of roots were measured at the indicated days after onset of Fe-deficiency treatment .(A) SPAD value of the newest leaves. (B) Fe(III)-chelate reductase activity. Non-transgenic line (NT), and transgenic rice expressing the (JPG)Click here for additional data file.S3 Fig(JPG)Click here for additional data file.S4 FigOsIRO2-overexpressing transgenic rice; Refre1, OsIRT1 promoter-refre1/372’ transgenic rice; 21 and 22, RI transgenic rice lines 21 and 22.NT, non-transformant; IRO2, (JPG)Click here for additional data file.S5 Figp < 0.05). NT, non-transformant; IRO2, OsIRO2-overexpressing transgenic rice; Refre1, OsIRT1 promoter-refre1/372’ transgenic rice; 21 and 22, RI lines 21 and 22.(A) Dry weight of straw per plant. (B) Dry weight of panicle per plant. Plants were harvested after 179 days of growth in calcareous soil, and the straw and panicle weight per plant was analyzed . Values followed by different letters were significantly different according by Tukey–Kramer’s HSD test ((JPG)Click here for additional data file.S6 Fig2 sub-lines of transgenic plants or NT plants) cultivated in calcareous soil. NT, non-transformant; IRO2, OsIRO2-overexpressing transgenic rice; Refre1, OsIRT1 promoter-refre1/372’ transgenic rice; 21 and 22, RI lines 21 and 22.(A) Number of panicles per plant. (B) Total number of grains per plant. (C) Total grain weight per plant. Each bar shows the value of a single plant (T(JPG)Click here for additional data file.S7 Fig(A) Plant height of individual lines. (B) SPAD value of the newest leaves of individual lines. (C) Value of plant height multiplied by the SPAD value of each plant used for line selection. Plant heights and SPAD values were measured at 21 DAT to calcareous soil. We selected Tachisugata-RI line 39 as it yielded the highest value of (C). A–E on the X-axis indicate non-transgenic Tachisugata plants. Numbers on the X-axis represent individual Tachisugata-RI lines. White circles on each line are data from individual plants. Red circles on the NT lines are the mean values of eight NT plants. Black circles are the mean values of eight plants of each Tachisugata-RI line. The red dotted line shows the mean values of all 40 NT plants.(JPG)Click here for additional data file.S8 Fig(A) Height of individual plants. (B) SPAD value of individual plants. TK-NT, Tsukinohikari non-transgenic plants; 22, Tsukinohikari-RI line 22; Ta-NT, Tachisugata non-transgenic plants; Ta-39, Tachisugata-RI line 39. Values for eight individual plants of each non-transgenic and transgenic line are shown in individual bars.(JPG)Click here for additional data file.


Furthermore, we find that MojV-G is antigenically distinct, indicating that MojV would less likely be detected in existing large-scale serological screening studies focused on well-established HNVs. Altogether, these data indicate a unique host-cell entry pathway for this emerging and potentially pathogenic HNV.In 2012, cases of lethal pneumonia among Chinese miners prompted the isolation of a rat-borne henipavirus (HNV), Mòjiāng virus (MojV). Although MojV is genetically related to highly pathogenic bat-borne henipaviruses, the absence of a conserved ephrin receptor-binding motif in the MojV attachment glycoprotein (MojV-G) indicates a differing host-cell recognition mechanism. Here we find that MojV-G displays a six-bladed β-propeller fold bearing limited similarity to known paramyxoviral attachment glycoproteins, in particular at host receptor-binding surfaces. We confirm the inability of MojV-G to interact with known paramyxoviral receptors  The attachment glycoprotein (HNV-G) of henipaviruses interacts with host receptors at the cell surface and is a major determinant of species tropism. Here the authors provide structural and functional evidence that the emergent henipavirus, Mòjiang virus, uses an entry mechanism that is independent of known paramyoxviral cellular receptors. Henipavirus, an emergent zoonotic group of bat-borne pathogens distributed throughout Asia, Australia, Central America and Africa23Three miners succumbed to severe pneumonia in Mòjiāng Hani Autonomous County, China, in June 2012. Investigation of bat, rat and musk shrew samples from within the cave, where the victims had most probably contracted the disease, led to the discovery of a novel rat-borne paramyxovirus, Mòjiāng virus (MojV)Asiatic Nipah virus (NiV) and Hendra virus (HeV) are prototypic members of the HNV genus and upon zoonotic spillover into human populations cause acute encephalitis and respiratory illness, with some reported case-fatality rates exceeding 90% refs , 3. Viru4471) and nectin-4 cell surface receptors123141516Paramyxovirus entry into a host cell requires two concerted steps: (i) viral attachment to a proteinaceous host cell surface receptor or sialic acid moiety and (ii) receptor-mediated fusion of the viral and host cell membranesDespite the diverse host receptors targeted, paramyxoviral HN, H and G proteins exhibit a conserved molecular architecture comprising an amino-terminal cytoplasmic tail, a single transmembrane helix, a stalk region and a carboxy-terminal six-bladed β-propeller18192021222327141729The capability of the paramyxoviral β-propeller to accommodate varied proteinaceous and glycan receptors provides a structural rationale for differential cellular and species tropism16F0 glycoprotein precursor to a disulfide linked, mature F1/F2 complex , MojV-G was crystallized and the structure was determined to 1.94 Å resolution , the β-propeller domain (residues 194–605) and the C-terminal extension (residues 606–625), was expressed in HEK 293T cells in the presence of an α-mannosidase inhibitor, kifunensine 3334. Con33solution .Paramyxovirinae attachment glycoproteins, with each blade of the propeller composed of four antiparallel β-strands , HN glycoprotein over 375 equivalent Cα atoms; PDB accession code 1V2I), Rubulavirus genus  and Avulavirus genus . Notably, MojV-G also poorly overlays with morbillivirus H glycoproteins (RMS deviation of 3.1 Å from measles virus haemagglutinin (MV-H), over 282 equivalent Cα atoms; 2RKC). Indeed, we observe that paramyxoviral receptor-interacting surfaces exhibit some of the greatest levels of structural deviation with respect to MojV-G. For example, the top face of the MojV-G β-propeller lacks the distinctive ephrin binding pocket observed in NiV-G  , and a tyrosine involved in neuraminidase activity and fusion promotion Y526 in NDV) in NDV26 (PHA-L) , whereas (PHA-L) . We suggThe spillover of novel pathogens from animals into human populations is unpredictable and constitutes an ever-persistent burden to global healthet al.The impact of MojV infection in humans remains a mystery. Rodent-borne MojV was discovered in a cave following the deaths of miners in Yunnan Province, Southern China. Although it is unknown whether MojV was directly responsible for these fatalities, sequencing of the MojV genome establishes a relationship with highly pathogenic HNVsGiven that HNV-G is a key determinant of host tropism25Paramyxoviridae. MojV-G is the only paramyxoviral glycoprotein to fall outside established receptor-specific structural groupingsN-linked glycosylation in the β-propeller domain  was PCR-amplified from codon-optimized cDNA  and cloned into the mammalian expression vector pHLsec1  to cleave glycosidic bonds of N-linked sugars within the di-N-acetylchitebiose core. Following deglycosylation, MojV-G purified by size-exclusion chromatography using a Superdex 200 10/300 GL column , in 10 mM TRIS pH 8.0, 150 mM NaCl buffer.The recombinantly produced, soluble MojV-G was purified by immobilized metal-affinity chromatography  at room temperature, using 250 mM imidazole for elution followed by size-exclusion chromatography using a Superdex 200 10/300 GL column , in 10 mM TRIS pH 8.0, 150 mM NaCl buffer. Purified protein was treated with EndoF−1) plus 100 nl precipitant at room temperature and appeared after 4–7 days in solution consisting of 16.4% (vol/vol) PEG 6000, 0.82 M lithium chloride and 0.082 M citrate pH 5.0. Crystals were flash frozen by immersion into a cryo-protectant containing 25% (vol/vol) ethylene glycol followed by rapid transfer into liquid nitrogen. X-ray diffraction data were recorded at beamline I24 (λ=0.96863 Å), Diamond Light Source, UK.Crystals were grown using a sitting drop vapour diffusion methodData were indexed, integrated and scaled with XIA2/XDS5457Structural alignments and structure-based phylogeny analysis were performed with the programme SHP61To produce anti-KV-G 1E10, BALB/c mice were sequentially immunized. The BALB/c mice were first genetically immunized by electroporation with mixtures of codon-optimized NiV matrix (M) protein, NiV-F and NiV-G expression plasmids, followed by NiV-M, HeV-F and HeV-G expression plasmids, and finally NiV-M, KV-F and KV-G expression plasmids. Vesicular stomatitis virus-ΔG pseudotyped with KV-F and KV-G (KVpp) was given via tail vein injection as the final protein boost before sacrifice and hybridoma production. Hybridomas were screened for anti-KV-G reactivity by both ELISA (using KVpp as the coated antigen) and flow cytometry of KV-G transfected cells.HEK 293T cells (ATCC CRL-3216) were transfected  with mammalian expression vectors encoding either MojV-G-HA, NiV-G-HA, HeV-G-HA, KV-G-HA or an empty vector control. At 48 h post transfection, cells were scraped, washed in 2% (vol/vol) fetal bovine serum (FBS)/PBS and incubated with neat supernatant from hybridoma cell lines expressing rabbit monoclonal antibodies 26, 45 6466Following the primary stain, cells were washed in 2% (vol/vol) FBS/PBS and incubated with a phycoerythrin (PE)-conjugated goat anti-rabbit polyclonal antibody (pAb)  diluted 1:1,000 or allophycocyanin-conjugated goat-anti-mouse pAb  diluted 1:1,000. Cells were washed in 2% (vol/vol) FBS/PBS, PBS and then fixed in 2% (vol/vol) PFA/PBS and subjected to flow cytometry (BD Cantoll). Cell surface expression of each G protein was assessed using a primary rabbit anti-HA pAb  at 1:1,000 dilution and a secondary PE-conjugated goat-anti-rabbit pAb  at 1:1,000 dilution. All incubations were performed on ice and all washes were carried out at 4 °C.HEK 293T (ATCC CRL-3216) and CHO pgsA-745 (ATCC CRL-2242) cells were transfected (using Lipofectamine LTX) with mammalian expression vectors encoding either MojV-G-HA, NiV-G-HA or an empty vector. At 48 h post transfection, cells were scraped, washed in 2% (vol/vol) FBS/PBS and incubated with either human ephrinB2-hFc, ephrinB3-hFc or hFc (R&D Systems) at 10 nM in 2% (vol/vol) FBS/PBS for 1 h. Cells were washed, stained with a goat F(ab’)2 anti-human IgG-PE  at a 1:1,000 dilution for 1 h in 2% (vol/vol) FBS/PBS, fixed and subjected to flow cytometry. EphrinB2 and B3 binding was adjusted for cell surface expression of each G protein by staining for a C-terminal HA epitope tag.HEK 293T cells were transfected with plasmids expressing MojV-G-HA or MV-H glycoprotein (as a positive control), scraped 48 h post transfection, washed in 2% (vol/vol) FBS/PBS and incubated with soluble CD150 engineered with a C-terminal FLAG epitope tag generously provided by Phillipe PlattetBSR-T7 (BHK-based cell line with stable expression of T7 polymerase)Gaussia luciferase. The target cells were trypsinized 12 h post transfection. The various target cells were allowed to recover in media in a falcon tube for 12 h, resuspended in media and equal amounts were applied to each well of the BSR-T7-transfected effector cells. Supernatant was harvested at 48, 72, 96 and 120 h post-application of target cells and assayed for Gaussia luciferase (NEB Gaussia luciferase assay system). This assay was modified to include (1) target cells, including CHO pgsA-745 (ATCC CRL-2242) cells stably expressing ephrinB2; (2) soluble NiV-G-hFc or hFc incubated with target cells for 1 h on ice at a concentration of 20 μg ml−1 before application of the cells to effector cells; (3) treatment of target cells with 20 μM kifunensine for 24 h before their application to effector cells; and (4) BSR-T7 effector cells transfected with heterotypic (in addition to homotypic) combinations of plasmids encoding F and G glycoprotein from a panel of HNV and HNV-like viruses.BSR-T7 cells (effector cells) were transfected (using Lipofectamine LTX) with expression plasmids encoding either the paired F and G glycoproteins, the F gene with pCAGGS or the G gene with pCAGGS across a panel of different HNVs. A549 (ATCC CCL-185), BHK-21 (ATCC CCL-10), U87 (ATCC HTB-14) and 293T (ATCC CRL-3216) target cells were transfected (using Lipofectamine LTX) with a plasmid encoding T7-driven Coordinates and structure factors of MojV-G have been deposited in the Protein Data Bank with the accession code 5NOP. The data that support the findings of this study are available from the corresponding authors upon request.How to cite this article: Rissanen, I. et al. Idiosyncratic Mòjiāng virus attachment glycoprotein directs a host-cell entry pathway distinct from genetically related henipaviruses. Nat. Commun.8, 16060 doi: 10.1038/ncomms16060 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Nature Communications8 Article: 14700 ; DOI: 10.1038/ncomms14700 (2017); Published 03132017; Updated 08142017The financial support for this Article was not properly acknowledged. The acknowledgements should include a reference to support, ‘by the ONR under award number N00014-16-1-3169 (Designing a Dynamic Platform that Provides Multiple Defense Mechanisms against Fouling)’ instead of ‘ONR under award number N00014-15-1-2157 (cell manipulation)’.


Tumor cells rely on aerobic glycolysis to generate ATP, namely the "Warburg" effect. 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, but its effect on cellular signaling pathways has not been fully elucidated. Herein, we sought to investigate the effect of 2-DG on ERK function in lung cancer cells. We found that 2-DG inhibits ERK phosphorylation in a time and dose-dependent manner in lung cancer cells. This inhibition requires functional LKB1. LKB1 knockdown in LKB1 wildtype cells correlated with an increase in the basal level of p-ERK. Restoration of LKB1 in LKB1-null cells significantly inhibits ERK activation. Blocking AMPK function with AMPK inhibitor, AMPK siRNA or DN-AMPK diminishes the inhibitory effect of 2-DG on ERK, suggesting that 2-DG—induced ERK inhibition is mediated by LKB1/AMPK signaling. Moreover, IGF1-induced ERK phosphorylation is significantly decreased by 2-DG. Conversely, a subset of oncogenic mutants of K-Ras, the main upstream regulator of ERK, blocks 2-DG—induced LKB1/AMPK signaling. These findings reveal the potential cross-talk between LKB1/AMPK and ERK signaling and help to better understand the mechanism of action of 2-DG. One of the primary hallmarks of cancer  is alterOf all the glycolysis inhibitors that have been evaluated, 2-deoxyglucose (2-DG) has been best characterized in animal models  and huma2-DG can efficiently inhibit cell growth and invasion, and potently facilitate apoptosis in various cancer cells ,18,19. HGCCAACGTGAAGAAGGAAATT. Lentiviral helper plasmids (pCMV-dR8.2 dvpr and pCMV-VSV-G) were obtained from Addgene, USA. Plasmids encoding wild-type (WT) LKB1 [Mouse monoclonal antibody against LKB1 (#ab15095) was purchased from Abcam, UK. Antibodies against total AMPK #2532), p-AMPKα Thr172 (#2535), p-ACC (phospho-acetyl-CoA carboxylase) Ser79 (#3661), total ERK1/2 (#9102), p-ERK1/2 Thr202/Tyr204 (#9101), total AKT (#9272), p-AKT Thr473 (#9271), p-S6K Thr389 (#9105) and Kras (#3965) were purchased from Cell Signaling Technology, USA. Rabbit polyclonal anti-actin antibody was purchased from Sigma-Aldrich, USA. Mouse anti-Ras antibody was purchased from Millipore, Germany. 2-DG, puromycin and IGF-1 were purchased from Sigma—Aldrich, USA. LY294002 (a PI3K inhibitor) was purchased from LC Laboratories. Compound C (an inhibitor of AMPK) solution was purchased from Calbiochem, USA. The lentiviral LKB1 short hairpin RNA (shRNA) construct and a negative control construct that was created in the same vector system (pLKO.1) were purchased from Open Biosystems, USA. LKB1 shRNA ID# is TRCN408, and its target sequence is WT) LKB1 , dominan32, p-AMPWT) LKB1  were con2.The LKB1 mutant  and LKB1 WT  NSCLC cell lines were purchased from the American Type Culture Collection . The isogenic K-Ras G13D and K-Ras WT HCT116 colon carcinoma cells were kindly obtained from Prof. Bert Vogelstein . Cells were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum  at 37°C in a humidified atmosphere with 5% CO6 293T cells were plated in 10-cm dishes. 24 hours later, cells were co-transfected with shRNA constructs (3 μg) together with pCMV-dR8.2 dvpr (3 μg) and pCMV-VSV-G (0.3 μg) helper constructs using Lipofectamine 2000 reagent. Two days later, viral stocks were harvested from the culture medium, which was filtered to remove non-adherent 293T cells. To select NSCLC cells that stably expressed shRNA constructs, cells were plated at subconfluent densities and infected with a cocktail of 1 ml of virus-containing medium, 3 ml of regular medium, and 8 μg/ml polybrene. Selection with 2 μg/ml of puromycin was started 48 hours after lentivirus infection. After about 4 weeks of selection, monolayers of stably infected pooled clones were harvested for use and cryopreserved. Western blot was used to evaluate LKB1 stable knockdown in NSCLC cells.Lentivirus stocks were prepared following the manufacturer’s protocol, as described previously . BrieflyThe GFP-LKB1 adenovirus was constructed as previously described . BrieflyTransfections were carried out with Lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen). Briefly, cells grown to 90% confluence in 6-well plates were transfected with 1.0 μg/well plasmids with 3 μl Lipofectamine 2000 and replaced with fresh growth medium after 4–6 hours.5’GAGUCUACAGUUAUACCAAtt-3’ and 5’-GCAUAUGGUUGUUCAUCGAtt-3’. Lamin A/C siControl (Santa Cruz Biotechnology) was used to control for any nonspecific off-target effects of siRNA transfection. Cells were transiently transfected with chemically synthesized AMPK siRNAs (200 pmol) or with the nonsilencing control siRNA using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Specifically, H522, H520, Calu-1 and H1650 cells were grown to 60–70% confluence in 6-well plates. Lipofectamine 2000 reagent was incubated with Opti-MEM1 for 5 min, and a mixture of siRNA was then added. After incubation for 20 min at room temperature, the mixture was diluted with medium and added to each well.SiRNA specific for AMPK-α1 (PRKAA1) and AMPK-α2 (PRKAA2) were purchased from Applied Biosystem (AB), with the corresponding sequences: Proteins were resolved by polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes . The membranes were blocked in Tris-buffered saline containing 0.2% Tween 20 and 5% fat-free dry milk and incubated first with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized with enhanced chemiluminescence detection reagent according to the manufacturer’s instructions .We previously demonstrated that 2-DG activates IGF1R signaling , thus weTo characterize the inhibitory effect of 2-DG on ERK phosphorylation in LKB1-WT cells, Calu-1, H522, H226, H520 and H1650 cells were treated with 2-DG (25 mM) for varying lengths of time. As shown in We previously demonstrated that 2-DG induces AKT phosphorylation in both LKB1 mutant and LKB1-WT NSCLC cells . AKT hasThe finding that 2-DG requires functional LKB1 to inhibit ERK in NSCLC cells  promptedNext, we sought to evaluate the impact of ectopic LKB1 expression on 2-DG—induced ERK activation in LKB1 null NSCLC cells. A549 and H23 cells were infected with adenovirus expressing GFP-LKB1 fusion protein or control adenovirus containing only GFP , and theAMPK, one of the canonical substrates of LKB1 as described above, has previously been shown to be potentially involved in regulating ERK signaling –31. To eTo further evaluate the role of AMPK in regulating ERK phosphorylation, LKB1 WT cells were transiently transfected with control siRNA or AMPKα siRNA, in the absence or presence of 2-DG. As shown in LKB1 WT cells were also transiently transfected with plasmids encoding control vector or dominant negative N157A AMPKα 1 (DN-AMPK). As shown in To investigate the effect of 2-DG on ERK activation induced by growth factors, LKB1 WT cells  were serum-starved for 4 hours and then pretreated with or without 2-DG before the addition of fetal bovine serum (FBS). As shown in We previously demonstrated that 2-DG induced ERK phosphorylation in three LKB1-WT cell lines, H1299, H1792 and H358  were treTo further evaluate the impact of various K-Ras mutations on 2-DG—induced LKB1/AMPK activation, HCT116 cells with only WT endogenous K-Ras were transfected with different cancer-derived K-Ras mutants, including 13D, 12V and 12D, and then were treated with 25 mM 2-DG for 2 hours. As shown in To confirm our findings in NSCLC cells, LKB1 WT/Kras WT H522 cells were transfected with plasmids encoding Kras G12V, WT Kras or vector, and then treated with 2-DG. As shown in Glucose is the major source of energy for cancer cells. Warburg demonstrated that cancer cells largely depend on aerobic glycolysis to generate ATP . A direcOne intriguing finding of our study is the potential cross-talk between LKB1/AMPK and ERK signaling. LKB1 is inactivated in 20–30% of NSCLC, ranking it as the third most frequently mutated gene in lung adenocarcinoma after p53 and K-Ras . As a tuMechanistically, our data indicate that LKB1 mediates this effect through the energy sensor AMPK . We provAnother intriguing finding of this study is that a subset of Ras mutations is capable of overriding 2-DG—induced LKB1/AMPK activation. Thus, it is conceivable that there is an interactive regulation between LKB1/AMPK and RAS-RAF-MEK-ERK signaling in cancer cells. Intriguingly, LKB1 loss is frequently accompanied by K-Ras mutations in NSCLC. In addition, concomitant loss of LKB1 significantly accelerates oncogenic K-Ras G12D-driven lung cancer progression in a mouse model . MoreoveOverall, 2-DG exerts inhibitory effects on the growth or survival of all lung cancer cells testedClick here for additional data file.


Using EdgeR, differentially expressed genes (DEG) were identified. Immune subset analysis was performed using Functional Analysis of Individual RNA-Seq/ Microarray Expression (FAIME) and immune gene RNA transcript count analysis. In total, 1,634 DEGs were identified, with a dominant immune signature observed in HPV(+) tumors. After normalizing the expression profiles to account for differences in B- and T-cell number, 437 significantly DEGs remained. A B-cell associated signature distinguished HPV(+) from HPV(−) tumors, and included the DEGs CD200, GGA2, ADAM28, STAG3, SPIB, VCAM1, BCL2 and ICOSLG; the immune signal relative to T-cells was qualitatively similar between TILs of both tumor cohorts. Our findings were validated and confirmed in two independent cohorts using TCGA data and tumor-infiltrating B-cells from additional HPV(+) HNSCC patients. A B-cell associated signal segregated tumors relative to HPV status. Our data suggests that the role of B-cells in the adaptive immune response to HPV(+) HNSCC requires re-assessment.Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has a better prognosis than it's HPV negative (HPV(−)) counterpart. This may be due to the higher numbers of tumor-infiltrating lymphocytes (TILs) in HPV positive (HPV(+)) tumors. RNA-Sequencing (RNA-Seq) was used to evaluate whether the differences in clinical behaviour simply reflect a numerical difference in TILs or whether there is a fundamental behavioural difference between TILs in these two settings. Thirty-nine HNSCC tumors were scored for TIL density by immunohistochemistry. After the removal of 16 TIL Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all cancers, with ~650,000 new cases reported and 350,000 HNSCC-related deaths per year worldwide , 2. Hist+ GZMA+ PRF1+ T-cells  as previously described [Frozen tumor sections taken immediately adjacent to the tissue analysed by RNA-Seq were stained with hematoxylin and eosin (H&E); tumors were assessed as TIL high (TILescribed  (Supplemescribed  (SupplemTwo HNSCC patient cohorts consisting of 137 HPV(+) and 407 HPV(−) tumors were analysed retrospectively for survival relative to TIL density and HPV status. The primary endpoint was death from HNSCC, i.e., disease-specific survival, as previously described [CD19 (B-cells) and CD4 and CD8A (T-cells) were used as the covariates. Unsupervised clustering of tumors was performed following variance stabilizing transformation of trimmed mean of M-values (TMM) normalized data. DEGs between HPV(+) and HPV(−) tumors were identified with a false discovery rate (FDR) corrected p-value of <0.05  and a fold change of >2 or <-2. A detailed description of the RNA-Seq data analysis performed can be found in the Supplementary Methods S3.RNA-Seq  was performed using the HiSeq 2000 platform  (Supplementary Methods S2). RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) under accession number GSE72536. RNA-Seq data was mapped using human genome (hg19) and TopHat (version 2.0.9), counted with HTSeq-count (version 0.5.4)  and diffGGA2), ADAM metallopeptidase domain 28 (ADAM28), CD200, Spi-B transcriptional factor (SPIB), stromal antigen 3 (STAG3), Vascular Cell Adhesion Molecule 1 (VCAM1), Inducible T-Cell Co-Stimulator Ligand (ICOSLG) and B-Cell CLL/Lymphoma 2 (BCL2) and adhere to the MIQE guidelines for RT-qPCR (Supplementary Methods S4) [STAG3 and CD200 was validated in the original tumor RNA, HPV(+) n=8 and HPV(−) n=8 patient tumor samples. B-cells (CD19+) isolated from an independent cohort of HPV(+) tumors (n=6) using a BD FACSAria™ sorter  were assessed for gene expression of ADAM28, BCL2, CD200, GGA2, ICOSLG, SPIB, STAG3, and VCAM1. RT-qPCR analysis was performed using the comparative Ct (cycle threshold) method (2-ΔΔCt) using Actin as the control gene and is defined as a normalized relative gene expression compared to a the control gene [Real-time quantitative reverse transcription PCR (RT-qPCR) assays were performed using Taqman® probes for golgi-associated, gamma adaptin ear containing, ARF binding protein 2 (hods S4) . Gene exrol gene .q-value <0.05) were identified with ConsensusPathDB [GO terms associated with biological processes and biological pathways that were significantly over-represented for DEGs  [+ and CD8+ T-cells  between the HPV(+) and HPV(−) cohorts.The distribution and proportions of TILs were assessed at both the tumor sample and group level. At the sample level, the gene expression (RNA-Seq) and surface protein expression (IHC) of  (FAIME)  was adaphttp://gdac.broadinstitute.org/runs/stddata_2015_11_01/data/HNSC/20151101/), the RNA-Seq methodology and processing have been described by TCGA [HNSCC RNA-Seq data (TCGA HNSC HiSeqV2 2015-02-24) was obtained from The Cancer Genome Atlas (TCGA) Genome Data Analysis Center (GDAC) Firehose website (


We previously demonstrated that monocyte-derived IL-4 DCs are more susceptible to HTLV-1 infection than autologous primary T-cells, suggesting that DC infection precedes T-cell infection. However, during blood transmission, breast-feeding or sexual transmission, HTLV-1 may encounter different DC subsets present in the blood, the intestinal or genital mucosa respectively. These different contacts may impact HTLV-1 ability to infect DCs and its subsequent transfer to T-cells. Using in vitro monocyte-derived IL-4 DCs, TGF-β DCs and IFN-α DCs that mimic DCs contacting HTLV-1 in vivo, we show here that despite their increased ability to capture HTLV-1 virions, IFN-α DCs restrict HTLV-1 productive infection. Surprisingly, we then demonstrate that it is not due to the antiviral activity of type–I interferon produced by IFN-α DCs, but that it is likely to be linked to a distinct trafficking route of HTLV-1 in IL-4 DCs vs. IFN-α DCs. Finally, we demonstrate that, in contrast to IL-4 DCs, IFN-α DCs are impaired in their capacity to transfer HTLV-1 to CD4 T-cells, both after viral capture and trans-infection and after their productive infection. In conclusion, the nature of the DCs encountered by HTLV-1 upon primo-infection and the viral trafficking route through the vesicular pathway of these cells determine the efficiency of viral transmission to T-cells, which may condition the fate of infection.Human T lymphotropic Virus type 1 (HTLV-1) is the etiological agent of Adult T cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Both CD4 + T-cell population. However, beside lymphocytes, HTLV-1 infects blood or monocyte-derived dendritic cells (DCs) in vitro. Moreover, we previously showed that DCs are more susceptible to HTLV-1 infection than autologous T-cells, suggesting that DCs might be the first cells to be infected upon primo-infection, and important intermediaries for the viral spread to surrounding lymphocytes. Interestingly, different DC subsets are found in the blood or in the mucosa, the two entry routes used by HTLV-1 during infection of new individuals. In this study, we show for the first time that the different DC subsets are not equally susceptible to HTLV-1 infection. Furthermore we demonstrate that DC subsets with an increased ability to capture HTLV-1 virion are restricted to HTLV-1 productive infection. This restriction is linked to DC maturation but not to the antiviral activity of IFN-α produced by resistant DCs. Finally, we demonstrate that efficient viral transmission to T-cells is dependent upon DC productive infection rather than trans-infection. Altogether, our results indicate that the nature of the DCs encountered by HTLV-1 upon primo-infection determines the efficiency of viral transmission to T-cells, which may condition the fate of infection.Human T-Lymphotropic Virus type 1 (HTLV-1) is the etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). In chronically infected patients, the provirus is mainly detected in the CD4 After in vivo ,9,10,11.[in vivo ,13 and t+ T-cells [in vivo experiments strongly suggest that infection of DCs is required for the establishment and maintenance of HTLV-1 infection in animal models. Consistent with these data, we recently showed that human monocytes-derived dendritic cells (MDDCs) are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro [in vivo.The role of DC infection in HTLV-1 dissemination to T-cells has been investigated in mice exposed to chimeric HTLV-1-infected cells, in which the HTLV-1 envelope had been replaced by that of the Moloney murine leukemia virus, to allow HTLV-1 to enter murine cells. In this context, DC depletion leads to a decreased proviral load in mouse CD4 T-cells . In addi T-cells . Altogetin vitro , supportIn addition to the blood, DCs are widely distributed in peripheral tissues and in mucosae, where antigen capture might occur. DCs play a central role in activating naïve T-cells and directing the subsequent immune response. DCs are constituted of several distinct subsets that differ in their ability to activate immunity or to promote tolerance . Since HWe therefore investigated whether different MDDC subsets that are similar to tissue-resident DCs exposed to HTLV-1 during primary infection in humans, are equally susceptible to HTLV-1 infection. Human MDDCs were generated with cytokines that allow differentiation into different subsets: interleukin-4 (IL-4) for myeloid DCs of the blood, transforming growth factor beta (TGF-β) for mucosal DCs of the gut and interferon-alpha (IFN-α) for inflammatory DCs found in injured skin . We demoThe French Comité de protection des personnes Ile de France has approved the use of human samples used in this study. CPP-IDF-2012-10-04SC to Dr. Gessain .2O), diphenyl iodonium chloride ; dynasore , methyl-β-cyclodextrin , nystatin , amiloride , latrunculin , cytochalasin D , genistein , wortmanin , rottlerin , Gö6953 , NSC23766 trihydrochloride  and IPA-3  were purchased from Sigma.Chlorpromazine -2 agonist ; TLR-5 agonist ; TLR-3 agonist (Poly(I:C); 10 μg/ml); TLR-4 agonist ; TLR-7/8 agonist  were purchased from Invivogen. Recombinant IFN-α (500–2000 IU/ml) was purchased from Tebu-Bio.The following antibodies were purchased from BD Biosciences: APC-H7-labeled mouse anti-Human CD14 (MφP9); V450-coupled mouse anti-Human CD11c (clone Ly6); PE-Cy7-coupled mouse anti-Human CD40 (clone 5C3); PerCPCy5.5-coupled mouse anti-Human HLA-DR (clone G46.6). APC-coupled mouse anti-Human DC-SIGN (DCN47.5) and Vio-Bright-FITC-coupled mouse anti-Human BDCA4 (clone AD517F6) were from Miltenyi. PE-coupled mouse anti-Human CD86 (clone IT2.2) was from Ebioscience. Mouse anti-HTLV-1 Gag p19 antibody (clone TP7) was from Zeptometrik, and biotin-coupled mouse anti-HTLV-1 Tax antibody  was pro2.Jurkat cells stably transfected with a plasmid encoding the luciferase (Luc) gene under the control of the HTLV-1 long terminal repeat (LTR) promotor (Jurkat-LTR-Luc)  were maiBlood was collected by Etablissement Français du Sang  from non-infected blood donors. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood on Ficoll density gradient. Monocytes were purified from PBMCs on Percoll gradients. Cells were cryopreserved in 10% dimethylsulfoxide (DMSO) - 50% FCS—40% culture medium before being used. Dendritic cells were generated after culture of monocytes in complete MDDC medium. IL-4 DCs were obtained after culture of monocytes in complete MDDC medium supplemented with GM-CSF and IL-4 for 5 days. Fresh cytokines were added 72 h later. When indicated, IL-4 DCs were further treated for 18 h by the addition of TLR agonists or recombinant IFN-α in the MDDC medium. TGF-β DCs were obtained after culture of monocytes in complete MDDC medium supplemented with GM-CSF, IL-4 and TGF-β for 5 days. Fresh cytokines were added 72 h later. IFN-α DCs were obtained from monocytes cultured in complete MDDC medium supplemented with GM-CSF and IFN-α for 3 days. Fresh cytokines were added 24 h later.5 cells) were co-cultured with C91-PL cells (2.5 x 104 cells), pre-treated with mitomycin C . MDDCs were then collected after 3h or 72h of co-culture, fixed and used for flow cytometry analyses  and imaging. When indicated, MDDCs were infected in presence of azido-thymidine  or treated with chloroquine  or diphenyl iodonium chloride  before co-culture with mitomycin-treated C91-PL cells.MDDCs (2.5 x 105 cells) were infected by biofilm preparation (100 μl) purified as previously described [When indicated, MDDCs (2.5 x 10escribed . At each5 cells) were plated in 48-well plates and treated with drugs targeting several endocytosis pathways for 3 h. Untreated cells were incubated in the same conditions and were used as controls. Treated and control MDDCs were then co-cultured with mitomycin-treated C91-PL cells (2.5 x 104 cells) for an additional 3 h. Cells were then fixed and stained for flow cytometry analyses.MDDCs (2.5 x 103 cells per well) were plated in Lab-Tek chamber slides (Nunc) previously treated with Poly-L-lysine  according to the manufacturer’s instructions and cultured for 18 h. When needed, LPS (1 μg/ml) was added. MDDCs were then co-cultured with mitomycin-treated C91-PL cells (ratio 10:1) for 3 h and fixed with 4% paraformaldehyde (PFA). Cells were extensively washed with PBS, quenched with NH4Cl solution (50 mM) and permeabilized with PBS—1% BSA—0.1% saponin. Cells were then stained using a serum from an HTLV-1 infected patient  followed by a DyLight 488-labeled Goat anti-Human IgG Antibody  and anti-CD82 antibody  followed by a DyLight 549-labeled anti-Mouse IgG Antibody . When indicated, cells were stained with anti p19Gag antibody followed by a DyLigh 549-labelled anti-mouse antibody and anti-human EE1A antibody  followed by a FITC-labelled anti-goat antibody (Dako). After washes in PBS and lastly in water, slides were mounted in Dapi-Fluoromount G (Southern Biotech). Samples were examined under a Leica spectral SP5 confocal microscope equipped with a 63x 1.4–0.6 oil-immersion objective using the LAS-AF software, or using a Zeiss LSM800 confocal microscope equipped with a 63x 1.4 oil immersion objective on the ZEN software. Images were analyzed using ImageJ.MDDCs  for 30 minutes. Cells were then extensively washed and fixed in 4% PFA. For lipid droplets staining, cells were fixed in 4% PFA, and stained with Nile Red  for 15 min. After several washes in PBS and lastly in water, slides were mounted in Dapi-Fluoromount G and fluorescence analyzed with routine inverted Zeiss axiovert 135 microscope. Fluorescent signals were quantified with ImageJ.5 cells) were collected, washed in PBS and stained with the following panel: APC-H7-labeled mouse anti-Human CD14; V450-coupled mouse anti-Human CD11c; PE-Cy7-coupled mouse anti-Human CD40; PerCPCy5.5-coupled mouse anti-Human HLA-DR, APC-coupled mouse anti-Human DC-SIGN, Vio-Bright-FITC-coupled mouse anti-Human BDCA4 and PE-coupled mouse anti-Human CD86. To analyze HTLV-1 capture, MDDCs were collected after co-culture with mitomycin-treated C91-PL cells, washed in PBS and fixed with 4% PFA. Cells were permeabilized with PBS—1% BSA—0.1% saponin, and stained with a mouse anti-p19gag antibody (1:1000) followed by a DyLight 488-coupled goat anti-mouse antibody (1:1000). Cells were then washed twice with PBS—1% BSA—0.1% saponin and once with PBS—1% BSA and finally surface-stained with V450-coupled anti-human CD11c antibody.MDDCs , fixed and permeabilized according to the manufacturer’s instructions (eBiosciences). Cells were stained with biotin-coupled anti-Tax antibody (LT4) followed by streptavidin labeled with PE-Cy7 . After extensive washes, cells were finally surface-stained with a V450-coupled anti-CD11c antibody. All results were acquired by flow cytometry using at least 50 000 events  and analyzed with FlowJo software.Genomic DNA was extracted using the Nucleospin blood kit  according to the manufacturer’s instructions. DNA concentration was determined with a NanoDropND1000 spectrophotometer (Thermo Scientific). Real-time quantitative PCR (qPCR) was performed on 10 ng of genomic DNA as previously described .5 cells) were plated in 48-well plates and treated with 500 IU/ml of recombinant IFN-α for 18 h. Cells were washed twice in phosphate-buffered saline (PBS) and total RNA was extracted using the RNeasy minikit . Extracted RNA was treated with RQ1-RNAse-free DNAse (Promega). RNA concentration was determined with a NanoDropND1000 spectrophotometer (Thermo Scientific). RT-PCR was performed on 500 ng of RNA using the SuperScript Reverse Transcriptase (Thermofisher). cDNA concentration was then determined with a NanoDropND1000 spectrophotometer (Thermo Scientific). cDNA amplification was performed on 200 ng of cDNA using the GoTaq polymerase (Promega) and Mx1 primers as previously described [IL-4 DCs (2.5 x 10escribed . PCR pro6 cells) were exposed to mitomycin-treated C91-PL cells (6 x 105 cells) for 4 h in 6-well plates. Then, DCs were magnetically isolated using the CD304 (BDCA-4/Neuropilin-1) Microbead kit . Isolated DCs (105 cells) were co-cultured with Jurkat-LTR-Luc (105 cells) in 48-well plates. Co-culture was performed for 48 h. Cells were then collected, and luciferase reporter activity assayed (Luciferase kit Promega). Results were normalized according to the amount of proteins determined by Bradford (Biorad).To determine the ability of MDDCs to transfer HTLV-1 without being infected, MDDCs (3 x 106 cells) were exposed to mitomycin-treated C91-PL cells (6 x 105 cells) for 4 h in 6-well plates. Then, DCs were magnetically isolated using the CD304 (BDCA-4/Neuropilin-1) Microbead kit. Isolated DCs (1 x 105 cells) were cultured in 48-well plates for 72 hours in the presence or absence of 100 μM of AZT added in culture medium each day. Jurkat-LTR-Luc cells (105 cells) were then added and the co-culture was maintained for 48 h. Cells were then collected, and luciferase reporter activity assayed. Results were normalized according to the amount of proteins determined by Bradford (Biorad).To determine the ability of productively infected MDDCs to transfer the virus to T-cells, MDDCs (3 x 104 cells) from the same donor were mixed with transduced Jurkat LTR-Luc cells (5 x 104 cells) and red-labelled C91-PL (5 x 103 cells), plated in Lab-Tek chamber slides previously treated with Poly-L-lysine and cultured for 45 minutes at 37°C. Cells were then fixed with 4% PFA for 15 minutes at room temperature. After several washes in PBS, slides were mounted in Dapi-Fluoromount G. Signals were acquired with an Axioimager Z1 microscope (Zeiss), and analyzed with ImageJ. Contacts between unstained MDDCs and GFP transduced Jurkat-LTR-Luc cells were manually counted from at least 10 pictures taken from the same slide.Jurkat LTR-Luc cells were transduced with Lenti-IRES-GFP as previously described . C91-PL 4 cells/well in 96-U-bottom-well plates and incubated for 24 h. Supernatant collected from MDDCs cultures (100 μl) or serial dilutions of recombinant IFN-α used for standard curve determination were added for an additional 17 h. Cells were then lysed and luciferase activity assayed.HL116 cells were seeded at 2 x 10One-way analysis of variance (ANOVA) with Bonferroni’s post-hoc multiple comparison test was used to determine statistically significant differences. Paired two-tail t-test was used to compare two groups from the same donor. Differences were considered significant if the p-value was < 0.05.gag viral protein over time (gag-positive cells significantly increased between 3h and 72 h post infection (p.i.), while it significantly decreased in IFN-α DCs, suggesting that IFN-α DC are not productively infected. Of note, in TGF-β DCs, the amount of p19gag-positive cells did not change significantly, suggesting a reduced level of infection. To confirm this result, the amount of proviral DNA was quantified 3 and 5 days after contact of the different DCs with purified HTLV-1 viral biofilm  that restrict viral cycle at different levels . FurtherMx-1 mRNA , caveolin-mediated endocytosis (methyl-β-cyclodextrin and nystatin), actin polymerization  or macropinocytosis (amiloride) before being co-cultured with C91-PL infected cells. We controlled that treatment with inhibitors did not change the viability of the different DC subsets compared to untreated DCs . Viral cg levels , suggestg levels , suggestg levels , suggestg levels . This fug levels , suggestg levels . AltogetSince HTLV-1 entry pathways, and intracellular localization are similar in immature IL-4 DCs that are susceptible to infection and in IFN-α DCs that are resistant to infection, we next wondered whether intravesicular pH could modify the ability of the virus to establish a productive infection. Indeed, previous studies reported that immature DCs have vesicles with neutral pH, while vesicles pH is more acidic in mature DCs . Using aWe have previously shown that productive infection of IL-4 DCs was the main route of HTLV-1 transmission to lymphocytes, although passive transfer from HTLV-1-exposed IL-4 DCs in presence of AZT may have occurred in rare cases . This paSurprisingly, AZT treatment of LPS-treated IL-4 DCs or IFN-α DCs also reduced luciferase signals observed with the untreated DC subsets, suggesting that the viral transfer observed with these subsets relies on their productive infection and not on long term viral capture . In addi+ T lymphocytes that represent the main infected cell population in chronically infected individuals, HTLV-1 proviral DNA is also detected in antigen presenting cells (APCs) such as myeloid DCs [in vitro MDDCs are more susceptible to HTLV-1 infection than autologous primary T-cells [In vivo, DCs come with different flavors and functions depending on their location in the body. Thus, virus interaction with DCs may have different outcomes depending on the nature of the DCs encountered in the course of infection. In this study, we used dendritic cells differentiated in vitro from human monocytes to generate different DC subsets mimicking those present in vivo. We demonstrate for the first time that DCs are not all equally susceptible to HTLV-1 infection. While immature DCs are more susceptible than tolerogenic-TGF-β DCs to HTLV-1 infection, inflammatory DCs and mature DCs are resistant to HTLV-1 infection.Apart from circulating CD4loid DCs , monocytloid DCs . We prev T-cells , supportSurprisingly, resistance is neither due to the presence of high level of type-I interferon produced by inflammatory DCs or mature DCs, nor to the exogenous recombinant interferon used to generate those DCs. Interestingly, these results are different from previous reports, including ours, obtained in T-cells, which showed that their susceptibility to HTLV-1 infection is sensitive to IFN-α ,27. Of nOur results also demonstrated that restriction was not due to a lack of viral entry in mature DCs, although the level of viral binding receptors is lower than in susceptible DCs. This suggests that viral capture by DCs that have been exposed to infected cells may use other receptors. Interestingly, HTLV-1 was observed in CD82 positive compartment in all DC subsets. CD82 is a tetraspanin receptor located in MVBs and at the plasma membrane, and has been shown to interact with the HTLV-1 envelope glycoprotein in T-cells . Thus, oWe showed here that maturation of DCs strongly limited their productive infection. Interestingly, it was shown that stimulation of primary hepatocytes with TLR agonists was able to inhibit replication of woodchuck hepatitis B virus (WHBV) in an interferon-independent pathway . In thisDC maturation is a complex process that modifies DC function such as antigen capture, antigen processing and presentation. Pathogen capture mainly occurs via macropinocytosis, a non-receptor mediated endocytic pathway. Macropinocytosis is a constitutive process in immature DCs , while iThese entry mechanisms may be consistent with the intracellular localization of HTLV-1 in the different subsets. Interestingly, earlier studies using electron microscopy analyses of MDDCs co-cultured with HTLV-1-infected lymphocytes, showed viral particles internalized in vesicles , althougThus, based on all our results, we propose a schematic model for HTLV-1 infection of DC subsets . When HTin vivo situation. The nature of the DCs encountered by HTLV-1 upon primo-infection, which display a differential susceptibility to HTLV-1 infection, would therefore determine the efficiency of viral transmission to T-cells. Finally, in contrast to the dissemination of HIV-1 by mature DCs, our results strongly suggest that the restriction of HTLV-1 infection in mature DCs and more importantly, their inability to transfer the virus to T-cells might be an efficient way for the host to limit HTLV-1 spread.Are these different outcomes of HTLV-1 infection in the different DC subsets important for the virus transfer to T-cells? In the HIV-1 model, DCs are resistant to HIV-1 infection due to the presence of several restriction factors , and thuS1 Fig(A) Cells were labeled with fluorochrome-coupled antibodies (black lines) directed against CD11c, HLA-DR, CD83 and CD40. Unstained cells were used as negative controls (black graphs). (B) IL-4 DCs infection was determined by the number of Tax-expressing cells among IL-4 DCs in presence or absence of AZT. Results from 3 independent experiments obtained from different donors are presented. Asterisks indicate statistically significant differences calculated using a paired t-test: *p<0.05.(TIF)Click here for additional data file.S2 Fig(A) IL-4 DCs were cultured for 18 h in the presence of exogenous IFN-α (2000 IU/ml). Untreated (white histogram) and IFN-α treated IL-4 DCs (light grey histogram) were stained with antibodies directed against CD11c, DC-SIGN, NRP-1, HLA-DR, CD86 and CD40. Unstained cells were used as negative controls (black graphs). (B) Treated and untreated IL-4 DC from the same blood donor were co-cultured with mitomycin-treated C91-PL cells for 3 h and the percentage of p19gag-positive DCs was assessed by flow cytometry. Results are representative of 3 independent experiments performed on different donors. T-test was performed to assess statistically significant differences: ns = non significant.(TIF)Click here for additional data file.S3 Fig(A) IL-4 DCs from same donor were cultured for 18 h with LPS (black dotted line) or with supernatant from LPS-matured DCs (light grey histogram) or left untreated as a control (black line). Cells were labeled with antibodies against CD11c, DC-SIGN, NRP-1, HLA-DR, CD86 and CD40. Unstained cells were used as negative controls (dark grey histogram). (B and C) IL-4 DCs, IL-4 DCs stimulated with supernatant from LPS-treated IL-4 DCs and LPS-treated IL-4 DCs from the same donor were co-cultured with mitomycin-treated C91-PL cells for 3 h (C) or 3 days (B) and the number of Tax-positive DCs (B) or the percentage of p19gag-positive DCs (C) were assessed by flow cytometry. DCs were discriminated from the C91-PL population by gating on CD11c expression. The results are shown as number of Tax-positive DCs (B) or as percentage of p19gag-positive DCs (C).(TIF)Click here for additional data file.S4 Fig(A) IL-4 DCs were left untreated (white histogram) or were treated for 18 h with the TLR-3 ligand PolyI:C, the TLR-4 ligand LPS, the TLR-7/8 ligand R848, the TLR-2 ligand PAM3CSK4 or the TLR-5 ligand flagellin (light grey histogram). Cells were labeled with antibodies against HLA-DR, CD86 and CD40. Unstained cells were used as negative control (dark grey histogram). (B) Supernatant from untreated IL-4 DC or DC treated with the TLR agonists were collected after 18 h of culture and type I IFN levels measured using HL116 reporter cells expressing ISRE-Luc.(TIF)Click here for additional data file.S5 FigGag (red) and EE1A (green) before analysis by confocal microscopy. Nuclei were counterstained with DAPI (blue). Representative images obtained by confocal microscopy are shown. Scale bars = 10 μm.IL-4 DCs, LPS-treated IL-4 DCs and IFN-α DCs were co-cultured with mitomycin-treated C91-PL cells for 3 h, and immunostained for p19(TIF)Click here for additional data file.S6 Fig(A) dynamin II inhibitors , clathrin-mediated endocytosis inhibitors , actin polymerization inhibitors  or macropinocytosis inhibitors  or (B) with constitutive macropinocytosis inhibitors , PI3K inhibitor , PKC inhibitors , Rac-1 inhibitors , Pak-1 inhibitor , and actin inhibitor . Cell viability was monitored by FACS using live-dead staining. (C) IL-4 DCs were left untreated (left panel) or treated (right panel) for 3 h with methyl B cyclodextrine (MβCD), fixed and incubated for 15 min with Nile red to stain lipid droplets (green). Nuclei were counterstained with DAPI (blue). Cells were visualized by fluorescence microscopy. (D-E) IL-4 DCs (black bars) or LPS treated IL-4 DCs (light gray bars) were treated for 3 h with nystatin or MβCD to deplete cholesterol (D) or nocodazole (Noco) or blebstatin (bleb) to block microtubule polymerization (E). After treatment, DCs were co-cultured with mitomycin-treated C91-PL cells for 3 h and viral capture determined by flow cytometry using p19gag staining. DCs treated with DMSO were used as controls. The percentage of DCs positive for p19gag was determined for each condition and normalized to that of the untreated control DCs. Results were obtained from 3 independent experiments performed with different blood donors.IL-4 DCs (black bars), LPS treated IL-4 DCs (light gray bars) or IFN-α DCs (white bars) were treated for 3 h with (TIF)Click here for additional data file.S7 Fig(A) Untreated or (B) LPS-treated IL-4 DCs were exposed to C91-PL for 4 h, left untouched  or magnetically isolated  and labeled with antibodies against CD11c, DC-SIGN, HLA-DR, CD86 and CD40. Unstained cells were used as negative controls (dark grey histogram). (C) The yield of C91-PL depletion after magnetic isolation of IL-4 DCs was estimated by flow cytometry using anti-human CD11c staining. The percentage of C91-PL cells (CD11c negative population) and the percentage of DCs (CD11c positive population) are indicated on each plot before (left) and after (right) magnetic isolation of DCs.(TIF)Click here for additional data file.