An HIV-1 vaccine continues to be a major target to halt the AIDS pandemic. The limited efficacy of the RV144 phase III clinical trial with the canarypox virus-based vector ALVAC and a gp120 protein component led to the conclusion that improved immune responses to HIV antigens are needed for a more effective vaccine. In non-human primates, the New York vaccinia virus (NYVAC) poxvirus vector has a broader immunogenicity profile than ALVAC and has been tested in clinical trials. We therefore analysed the HIV immune advantage of NYVAC after removing viral genes that act on several signalling pathways , as well as genes of unknown immune function. We generated a series of NYVAC deletion mutants and studied immune behaviour (T and B cell) to HIV antigens and to the NYVAC vector in mice. Our results showed that combined deletion of selected vaccinia virus (VACV) genes is a valuable strategy for improving the immunogenicity of NYVAC-based vaccine candidates. These immune responses were differentially modulated, positive or negative, depending on the combination of gene deletions. The deletions also led to enhanced antigen- or vector-specific cellular and humoral responses. These findings will facilitate the development of optimal NYVAC-based vaccines for HIV and other diseases. Vaccine development remains among the best hopes for controlling the HIV/AIDS pandemic.UNAIDS (Joint United Nations Programme on HIV/AIDS) estimates that at the end of 2016, 36.7 million people worldwide lived with HIV  ,3. High 1% ,3. HiAs an alternative to the ALVAC vector, the highly attenuated vaccinia virus (VACV) strain NYVAC  is a potB8R and/or B19R  [A46R [A52R, K7R and B15R [In mice, we were able to improve HIV-1-specific immune responses to the NYVAC-C recombinant vector expressing HIV-1 antigens by deletion of immunomodulatory genes such as ctively) , by singy) [A46R , or by sand B15R ,17.B8R. We thus characterized HIV- and VACV-specific CD4 and CD8 T cells and antibody levels in mice immunized with these NYVAC deletion mutants. The deletions modulated HIV-1- and VACV-specific responses distinctly and some provided more immune enhancement than others. These results demonstrate that NYVAC-based vaccine immunogenicity can be regulated by genetic modification of the parental vector, which could extend use of these vectors for vaccine development.To further describe the role of viral genes in NYVAC vector immunogenic potential, we sought to define in mice the effect of deleting from the NYVAC-C genome various combinations of viral genes that inhibit TLR, IFN and cytokine/chemokine host-cell antiviral pathways, as well as some unknown non-essential genes that accompanied Animal experimental protocols were approved by the Ethical Committee of Animal Experimentation (CEEA-CNB) of Centro Nacional de Biotecnologia  in accordance with Spanish National Royal Decree (RD 1201/2005), International EU Guidelines 2010/63/UE on protection of animals used for experimentation and other scientific purposes and Spanish National Law 32/2007 on animal welfare and their exploitation, transport and sacrifice .l-glutamine  and 10% new-born calf serum  for BSC-40 cells or 10% foetal calf serum  for CEF. Cells were maintained in humidified air with 5% CO2 at 37 °C. Poxvirus strains used included the genetically attenuated vaccinia-based vector NYVAC-WT , the recombinant NYVAC-C expressing gp120 as a cell released product and Gag-Pol-Nef from the clade C CN54 HIV-1 isolate as an intracellular polyprotein [African green monkey kidney cells (BSC-40) and primary chicken embryo fibroblasts (CEF) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin , 2 mM yprotein   from the NYVAC-C genome, was obtained by sequential cloning of B6R and B10R recombination flanking sequences into the plasmid pGem-Red-GFP wm [B6R gene with oligonucleotides LFB6R-AatII-F (5′-GGAATGACGTCCTCCCAATATGTG-3′) (AatII site underlined) and LFB6R-XbaI-R (5′-GCTCTAGACTCAATTCATTCTAGC-3′) (XbaI site underlined). The left flank was digested with AatII and XbaI and cloned into plasmid pGem-Red-GFP wm previously digested with the same restriction enzymes to generate pGem-RG-LFsB6R wm (4881 bp). The right flank of the B10R gene was amplified by PCR from the NYVAC genome with oligonucleotides RFB10R-ClaI-F (5′-CCATCGATTTGAAAATGAAAATATAAATAG-3′) (ClaI site underlined) and RFB10R-BamHI-R (5′-CGGGATCCAGTAGATATGATCTATATTC-3′) (BamHI site underlined), digested with ClaI and BamHI and inserted into the ClaI/BamHI-digested pGem-RG-LFsB6R wm to generate pGem-RG-LFsB6R-RFB10R wm (5225 bp). The repeated left flank of the B6R gene was amplified by PCR from the NYVAC genome with oligonucleotides LFB6R′-EcoRI-F (5′-CGGAATTCCTCCCAATATGTGTACG-3′) (EcoRI site underlined) and LFB6R′-ClaI-R (5′-CCATCGATCTCAATTGATTCTAGC-3′) (ClaI site underlined), digested with EcoRI and ClaI and inserted into the EcoRI/ClaI-digested pGem-RG-LFsB6R-RFB10R wm. The resulting plasmid, pGem-RG-B6R-B10R-wm (5558 bp), was confirmed by DNA sequence analysis and directs deletion of the B6R-B10R cassette from the NYVAC genome.The plasmid transfer vector pGem-RG-B6R-B10R-wm, used for deletion of d-GFP wm . The NYVA52R, K7R and B15R ORFs from the NYVAC-C genome, respectively, were obtained by the same method and have been reported [The plasmid transfer vectors pGem-RG-A52R-wm, pGem-RG-K7R-wm and pGem-RG-B15R-wm, used to delete reported .6) were infected with 0.005 pfu (plaque-forming units)/cell of parental virus and transfected 1 h later with 6 μg DNA of specific plasmid transfer vector using Lipofectamine . At 72 h post-infection, cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. Deletion mutants were selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells, during which plaques were screened for Red2/GFP fluorescence. In the first three passages, viruses from selected plaques expressed both fluorescent proteins; in the next two passages, viral progeny from selected plaques expressed only one fluorescent marker. In the last two passages , viruses from selected plaques did not express a fluorescent marker due to marker loss by homologous recombination within the repeated flanking DNA sequences.The different NYVAC-based deletion mutants generated and the corresponding parental viruses and plasmid transfer vectors used in the infection/transfection protocol are listed in To test for correct generation and purity of the deletion mutants, viral DNA was extracted from BSC-40 cells infected at 5 pfu/cell with NYVAC-WT, NYVAC-C, or the different NYVAC-C deletion mutants. Cell membranes were disrupted by proteinase K treatment  pH 8, 100 mM NaCl, 1% SDS; 1 h, 55 °C), followed by incubation with RNase A (80 μg/mL). Viral DNA was precipitated using 2-propanol. Different sets of primers annealing in the gene-flanking regions to be deleted were used for PCR analysis of the loci. The amplification reactions were carried out with Phusion High-Fidelity DNA polymerase . Primers used and size of the expected PCR products are shown in To test correct expression of HIV-1 antigens by the deletion mutants, monolayers of BSC-40 cells were infected at 5 pfu/cell with NYVAC-WT, NYVAC-C, or the different NYVAC-C deletion mutants. At 24 h post-infection, cells were lysed in Laemmli buffer; cell extracts were fractionated by 8% SDS-PAGE and analysed by Western blot using rabbit polyclonal anti-gp120 antibody  or rabbit polyclonal anti-gag p24 serum  to evaluate gp120 and Gag-Pol-Nef (GPN) protein expression, respectively. Anti-rabbit-horseradish peroxidase  was used as secondary antibody. Immune complexes were detected by enhanced chemiluminescence .2). At different times post-infection , cells were removed by scraping and freeze-thawed three times; lysates were prepared from 5 × 105 cells/mL and briefly sonicated. Virus titres in cell lysates were determined by immunostaining in a plaque assay in BSC-40 cells, as described [To determine virus growth profiles, CEF monolayers grown in 12-well tissue culture plates were infected at 0.01 pfu/cell with NYVAC-WT, NYVAC-C, or the different NYVAC-C deletion mutants. Following virus adsorption , the inoculum was removed. Infected cells were washed once with serum-free DMEM and incubated with fresh DMEM containing 2% FCS  and HIV-1 CN54Gag-Pol-Nef (GPN) fusion protein (pcDNA-CN54GPN) have been reported [The two DNA constructs expressing the HIV-1 reported . Plasmid140–148 , described as an immunodominant epitope in BALB/c mice [The HIV-1 peptide pools Env-1, Env-2, Gag-1, Gag-2, GPN-1, GPN-2, GPN-3 and NEF were provided by the EuroVacc Foundation  and have been described . They spB/c mice , was usen = 4) received 100 μg DNA-C (50 μg pcDNA-CN54gp120 + 50 μg pcDNA-CN54GPN) by the intramuscular route (i.m.); two weeks later, they received an intraperitoneal (i.p.) inoculation of 1 × 107 pfu of the corresponding virus. The control group was primed with sham DNA (DNA-ϕ) and boosted with non-recombinant NYVAC-WT. At 53 days after the last immunization (memory phase), mice were sacrificed and spleens and sera were processed for intracellular cytokine staining (ICS) and Enzyme-Linked ImmunoSorbent (ELISA) assays to analyse cellular and humoral immune responses to HIV-1 and VACV antigens, respectively. Representative data are shown for 2–3 experiments.BALB/c mice were purchased from Harlan. The DNA prime/poxvirus boost immunization protocol was used to assay the immunogenicity of the deletion mutants. Groups of 6- to 8-week-old female mice (6 splenocytes (erythrocyte-depleted) were seeded on 96-well plates and stimulated (6 h) in complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FCS, 1 μL/mL Golgiplug  and 1 μg/mL of the different HIV-1 peptide pools or 10 μg/mL of E3 peptide. After stimulation, cells were washed, stained for surface markers, permeabilized  and stained intracellularly using appropriate fluorochromes. Fluorochrome-conjugated antibodies were used for functional analyses  and for phenotypic analyses . Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen). All antibodies were from BD Biosciences. Cells were acquired on an LSRII flow cytometer . Data analyses were performed using FlowJo software v.8.5.3 . The number of lymphocyte-gated events ranged between 105 and 106. After gating, Boolean combinations of single functional gates were generated using FlowJo to determine the frequency of each response, based on all possible combinations of cytokine expression or of differentiation marker expression. Background responses in negative control samples were subtracted from those in stimulated samples for each functional combination. Graphs were generated using GraphPad software .The magnitude and phenotype of HIV- or VACV-specific T cell responses were analysed by ICS. After an overnight rest, 4 × 10450 value of the naïve control.Antibody binding to Env and vaccinia virus proteins in serum was assessed by ELISA as described . Sera fr50  values represent the reciprocal of the serum dilution giving 50% reduction in plaque number compared with virus incubated without serum.To measure the neutralizing titre of anti-VACV antibodies, vaccinated mice were exsanguinated and sera were prepared and heated at 56 °C for 30 min to inactivate complement. Two-fold dilutions of serum in DMEM  supplemented with 2% NCS were prepared and incubated with about 400 pfu of purified VACV WR for 1 h at 37 °C before plaque assay on BSC-40 cells grown in 6-well plates. After 48 h of incubation at 37 °C, plaques were visualized by staining with 1% crystal violet in 2% ethanol. NDn = 5, 6–8 weeks old) were infected intraperitoneally (i.p.) with 107 pfu of NYVAC-WT, NYVAC-C or NYVAC-C deletion mutants in TLR immunomodulatory genes. PBS-treated mice were used as control group. One month later, animals were challenged intranasally (i.n.) with 5 × 106 pfu of wild-type VACV WR. The weight loss was monitored daily for 15 days.Female BALB/c mice  and calculated confidence intervals and is tests ,31. OnlyA46R, A52R, K7R and B15R; B8R and/or B19R and/or B6R-B10R cassette). Western blot analysis indicated that NYVAC-C-based deletion mutants expressed HIV-1 gp120 (120 kDa) and GPN (150 KDa) proteins at levels similar to the parental NYVAC-C virus . Mice primed with sham DNA (DNA-ϕ) and boosted with non-recombinant NYVAC-WT were used as controls. A head-to-head comparison of immunogenicity among different NYVAC vectors is described below.To define the effect of the distinct viral gene deletions in NYVAC-C vector immunogenicity, we used a mouse immunization protocol based on a DNA prime/poxvirus boost regime. This protocol is more immunogenic in activating T cell responses to HIV-1 antigens than the homologous combination of viral vectors and is being tested in clinical trials ,11. BALBA46R, which encodes an inhibitor of TLR signalling, enhances immunogenicity of the NYVAC-C recombinant virus [A52R, K7R and B15R genes from a recombinant virus that already lacked A46R (NYVAC-C-∆TLR1), to generate deletion mutants NYVAC-C-∆TLR2 (∆A46R/∆A52R), NYVAC-C-∆TLR3 (∆A46R/∆A52R/∆K7R) and NYVAC-C-∆TLR4 (∆A46R/∆A52R/∆K7R/∆B15R). In DNA-C-primed mice boosted with the different viruses, the breadth of the HIV-1-specific responses was similar in all groups. CD4+ T cell responses were directed mainly against the Env pool, whereas CD8+ T cell responses were distributed between Env and GPN pools. With the exception of NYVAC-C-∆TLR3 virus, the deletion mutants triggered an HIV-1-specific memory response mediated mainly by CD8+ T cells ; single deletion of A46R or additional combined deletions (K7R and B15R) did not improve this response. The HIV-1-specific memory CD4+ T cells elicited by NYVAC-C and the deletion mutants had mainly an effector memory (EM) phenotype , double (NYVAC-C-∆TLR2) and triple (NYVAC-C-∆TLR3) gene deletion mutants induced greater magnitudes of CD4henotype A, right.+ T cells, the NYVAC-C-∆TLR1, -∆TLR2 and -∆TLR4 deletion mutants induced significantly larger HIV-1-specific responses than parental NYVAC-C, with respective increases of 3.7-, 2.7- and 2.9-fold. In this cell subset, single deletion of the A46R gene yielded the highest HIV-1-specific CD8+ T cell response; additional deletions did not increase these values. Deletion of the K7R gene in NYVAC-C-∆TLR2 (NYVAC-C-∆TLR3) reduced CD8+ T cell responses, which recovered after B15R deletion (NYVAC-C-∆TLR4). In all groups, the HIV-1-specific memory CD8 T cells elicited were distributed between the EM and TEM phenotypes. For parental NYVAC-C, distribution of the response was 60% EM/40% TEM. This ratio was maintained in the NYVAC-C-∆TLR1 group but was reversed to 40% EM/60% TEM in NYVAC-C-∆TLR2 and NYVAC-C-∆TLR3 mutants. For NYVAC-C-∆TLR4, the response was distributed almost equally between the two phenotypes.For CD8+ T cell responses, by about 1.5-fold. All deletion mutants nonetheless induced higher anti-VACV responses than parental NYVAC-C  and double (NYVAC-C-∆TLR2) mutants. For anti-vector immunity, the sequential removal of three or four VACV-TLR inhibitors was necessary to enhance responses.B6R, B9R and B10R) with IFN—(B8R and B19R) and cytokine—(B7R) VACV inhibitors increased NYVAC-C immunogenicity. We deleted the B6R-B10R ORF cassette from a recombinant virus that lacked B19R (NYVAC-C-∆B19R), to generate NYVAC-C-∆B19R/∆B6R-B10R. Using the DNA prime/poxvirus boost approach, we evaluated the immune response induced by the resulting virus compared with parental NYVAC-C and with the single (NYVAC-C-∆B19R) and double (NYVAC-C-∆B8R/∆B19R) mutants.Since deletion of VACV genes that block IFN type I and II pathways improves the immunogenicity of NYVAC-C recombinants in mice , we expl+ T cell responses (~1.2-fold), while values for NYVAC-C-∆B8R/∆B19R virus were similar to NYVAC-C  and B19R cytokine inhibitor did not alter these values. In all groups, the HIV-1-specific memory CD8+ T cells were distributed mainly between the EM and TEM phenotypes. After NYVAC-C boost, approximately 60% and 40% of the HIV-1-specific response were EM and TEM, respectively, whereas the ratio in the NYVAC-C-∆B19R and NYVAC-C-∆B19R/∆B6R-B10R groups was 25% EM/75% TEM. For NYVAC-C-∆B8R/∆B19R, the response was distributed almost equally between the two phenotypes.For CD8+ T cell responses were enhanced in NYVAC-C-∆B8R/∆B19R and NYVAC-C-∆B19R/∆B6R-B10R groups, with ~2-fold increases, whereas for parental NYVAC-C and the single-gene deletion mutant NYVAC-C-∆B19R, VACV-specific responses were of similar magnitude  only increased the anti-Env response at low serum dilutions (1:100). The highest antibody response was observed after sequential deletion of A46R and A52R genes (NYVAC-C-∆TLR2). We found that only NYVAC-C-∆TLR3 and NYVAC-C-∆TLR4 mutants improved the humoral anti-vector response induced by wild-type NYVAC virus, although the deletion mutants elicited higher anti-VACV antibody levels than parental NYVAC-C  and double deletion of A46R and A52R genes (NYVAC-C-∆TLR2) induced the lowest anti-vector humoral and neutralizing responses after a single i.p. immunization; however, animals were protected. To determine to what extent single and sequential gene deletion of VACV-TLR inhibitors impact on the vaccine efficacy compared with the NYVAC-C parental virus, mice were vaccinated i.p. and challenged i.n. one month later with a lethal dose of wild-type VACV WR. As shown in Given the modest efficacy observed in the RV144 clinical trial, the scientific community has focused on generating and optimizing vaccine candidates with improved immunogenicity, able to confer higher protection.https://clinicaltrials.gov/). Despite the safety and immunogenicity profiles of these attenuated VACV strains, it would be desirable to develop more efficient vectors that enhance the magnitude, breadth, polyfunctionality and durability of the T and B cell immune responses to exogenously expressed antigens. Various strategies are being used to achieve this purpose. One is deletion of viral immunomodulatory genes still present in the vector genome, whose products are predicted to interfere with optimal induction of cellular and humoral immune responses to vector-expressed antigens. We previously reported enhanced immunogenicity of MVA- and NYVAC-based recombinants with single, double or multiple deletions of VACV immunomodulatory genes such as C12L [C6L and/or K7R [A41L and/or B16R [F1L [B8R and/or B19R [A46R [A52R, K7R and B15R in combination [Poxviruses and particularly the highly attenuated VACV strains such as MVA and NYVAC, are being widely tested for potential HIV vaccines and are components in some of the clinical trials planned in the next few years ,33,34  in response to HIV antigens and to the VACV vector  and the HIV-1 clade C polyprotein Gag-Pol-Nef and tested them in mice for their immunogenic characteristics  or combined with deletion of an essential viral replication gene (udg) (MVA∆5) in the genome of a recombinant MVA vector that expressed HIV gag and env genes [udg gene did not further improve HIV responses, however and offset the enhancement of vector-specific antibody titres due to immunization with the parental virus (∆4). Holgado et al. similarly reported that simultaneous deletion of A44L, A46R and C12L genes improved both innate and adaptive VACV-specific T cell immune responses in immunized mice, although the effect of these deletions in immunogenicity for a heterologous antigen was not tested [Other groups have analysed the effect of multiple deletions in immunomodulatory genes on immune responses in the context of another attenuated poxvirus strain, MVA. Garber et al. assayed the effect on antigen-specific immune responses of simultaneous deletion of nv genes . Followit tested . Using MAlthough the results for MVA could not be extrapolated to NYVAC because of differences in vector genomes, it seems clear that combined deletion of selected immunomodulatory genes in the NYVAC genome should be considered for the design of viral recombinants as vaccine candidates.


Short chain fatty acids (2 to 6 carbons in length) are ubiquitous lipids that are present in human plasma at micromolar concentrations. In addition to serving as metabolic precursors for lipid and carbohydrate synthesis, they also act as cognate ligands for two known G protein-coupled receptors (GPCRs), FFAR2 and FFAR3. While there is evidence that these receptors may inhibit the progression of colorectal cancer, their roles in breast cancer cells are largely unknown. We evaluated the effects of enforced overexpression of these receptors in two phenotypically distinct breast cancer cell lines: MCF7 and MDA-MD-231. Our results demonstrate that both receptors inhibit cell invasiveness, but through different signaling processes. In invasive, mesenchymal-like MDA-MB-231 cells, FFAR2 inhibits the Hippo-Yap pathway and increases expression of adhesion protein E-cadherin, while FFAR3 inhibits MAPK signaling. Both receptors have the net effect of reducing actin polymerization and invasion of cells through a Matrigel matrix. These effects were absent in the less invasive, epithelial-like MCF7 cells. Correspondingly, there is reduced expression of both receptors in invasive breast carcinoma and in aggressive triple-negative breast tumors, relative to normal breast tissue. Cumulatively, our data suggest that the activation of cognate receptors by short chain fatty acids drives breast cancer cells toward a non-invasive phenotype and therefore may inhibit metastasis. Among these, FFAR1 and FFAR4 are activated by long and medium chain fatty acids (LCFA/MCFA) such as oleate and palmitate, while FFAR2 and FFAR3 are activated by short chain fatty acids (SCFA) such as propionate and butyrate [Metastasis is a multi-stage process involving genetically unstable tumor cells that undergo phenotypic changes that allow them to egress from the primary tumor and colonize a distant tissue site with a favorable microenvironment , 2. Meta1,3 –17, LPA11,3 [1-6 , 19, and1 & EP4) , 21. The1 & EP4) , 23. Thebutyrate –25. FFARbutyrate , leptin butyrate , productbutyrate , glucosebutyrate , and secbutyrate . Similarbutyrate , 30–33. butyrate , 34. TakPrevious studies suggest that FFAR2 and FFAR3 are involved in the regulation of cancer development. This led us to investigate the mechanisms underlying FFAR2- and FFAR3-mediated effects on breast cancer cells, particularly pertaining to metastasis. In this study, we report that expression of FFAR2 and FFAR3 regulates YAP1 phosphorylation and that FFAR2 also regulates E-cadherin in MDA-MB-231 cells. Furthermore, through siRNA knockdown studies we identify LATS1, an upstream regulator of YAP1, as an obligate mediator of the E-cadherin changes observed in FFAR2 expressing MDA-MB-231 cells. Unlike FFAR2, FFAR3-mediated YAP1 elevation does not correlate with E-cadherin levels, which is likely to be regulated instead by the MAPK/ERK1/2 pathway.Primary antibodies: E-cadherin , total YAP1 (Santa Cruz #G2710), Phospho-YAP1 S127 , LATS1 , p44/42 MAPK , Phospho-p44/42 MAPK , β-actin antibody (Sigma #A 5316), and V5 tag antibody (ThermoFisher Scientific #R960-25). Secondary antibodies: Horseradish peroxidase-conjugated goat anti-rabbit  and anti-mouse , Alexa Fluor 594 (ThermoFisher Scientific #A11005) and Alexa Fluor 488 phalloidin (ThermoFisher Scientific #A12379).Reagents: Propionate (propionic acid # 402907) from Sigma-Aldrich Corporation.MDA-MB-231 (ATCC #HTB-26), MCF-7 cells (ATCC #HTB-22), HEK293 cells (ATCC #CRL-1573) and HEK293T cells (ATCC #CRL-3216) were maintained as monolayer cultures on tissue culture dishes at 37°C, 5% CO2, 100% humidity in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS)and antibiotics.5’-TGGGTCTGGTCTTTGGGTTG-3’ and the reverse primer 5’-CACGTAGCAGAAGGCTGTGA-3’ and FFAR3 forward primer 5’- AACCTCACCCTCTCGGATCT and reverse primer 5’- GCCGAGTCTTGTACCAAAGC, the amplified product was inserted into pcDNA3.1/V5-His TOPO expression vector according to manufacturer’s instructions.FFAR2-V5 and FFAR3-V5 were constructed by amplifying the FFAR2/FFAR3 coding DNA sequence (CDS) utilizing FFAR2 forward primer To generate MCF-7 and MDA-MB-231 cell lines stably overexpressing either empty vector pcDNA3.1/V5-His (referred to as vector control) or the FFAR2-V5 and FFAR3-V5 (referred to as FFAR2- and FFAR3-overexpressing), respective plasmid constructs were linearized with restriction enzyme PvuI-HF following which they were transfected into the cells with Lipofectamine 3000 (Thermo Fisher #L3000015). The transfected cells were cultured in 1 mg/ml G418 to select for stable transfectants.5’-UUCUCCGAACGUGUCACGU-3’) or target-specific (5’-CAUACGAGUCAAUCAGUAA-3’) siRNA using Lipofectamine 3000 reagent per manufacturer’s instructions. Cells were incubated for 48 hours after transfection, and processed for western analysis.For LATS1 knockdown studies, the indicated cells were transfected with 75 pmol of control  cells were seeded onto the top chamber of a Matrigel invasion chamber (Corning #354480) in serum-free media and the lower part of the chamber was in contact with 10% FBS containing culture media. The treatment where mentioned was carried out by adding 1 mM propionate diluted in plain DMEM to the upper chamber and culture media with 10% FBS was added to the lower chamber. Following a 24-hour incubation, the upper surfaces of the transwell chambers were gently wiped with cotton swabs and the invading cells were fixed in methanol, stained with crystal violet solution, washed, and air dried. The invading cell numbers were counted in five randomly selected microscope fields (20×).The escribed . Briefly-ΔΔCT method as described [mRNA quantification was performed essentially as described . Total Rescribed .This assay was conducted essentially as described , 41. BriTo understand the functions of FFAR2 and FFAR3 in breast cancer metastasis, we evaluated two breast cancer cell lines that have contrasting EMT phenotypes reflected by their EMT score: 1.0 = highly mesenchymal, -1.0 = highly epithelial . MCF-7 cCDH1, the gene encoding E-cadherin, was significantly upregulated in FFAR2-expressing cells relative to control cells and FFAR3-expressing cells , which has a gene profile of 1992 breast carcinoma samples and 144 normal breast tissues [FFAR2 and FFAR3 gene expression was significantly downregulated in invasive  [www.proteinatlas.org) [Since our data suggest that FFAR2 and FFAR3 inhibit EMT, we speculated that expression of these receptors is likely to be reduced in invasive breast cancer tissues. Therefore, to determine the expression pattern of the SCFA receptors in breast tumors, we made use of Curtis breast dataset available on the Oncomine database  . Expresslas.org) . We obseCDH1 (E-cadherin) expression specifically in mesenchymal-like MDA-MB-231  in breast cancer metastasis and the mechanism underlying this process. Since metastasis depends in part on the activation of EMT , we evalA-MB-231  and MDA-A-MB-231 , while nA-MB-231  or BT-47A-MB-231 . In contA-MB-231 . This suin vitro manipulation, we evaluated the relative expression patterns of FFAR2 and CDH1. If FFAR2 activity has a functional impact on the regulation of E-cadherin we would expect a positive correlation between these two genes. Indeed, the relationships are positive and highly significant in both cell lines and tumor samples  No immunoreactivity was observed in pcDNA3.1-containing cells. (C-D) FFAR2- and (E-F) FFAR3-containing cells demonstrate robust labeling that is largely restricted to the plasma membrane.(TIF)Click here for additional data file.S2 Fig50 values of 7 and 10 μM for FFAR2 and FFAR3, respectively.Propionate was prepared by diluting in DMEM to the indicated concentrations and used to treat HEK293 cells transfected with pcDNA3.1 or the indicated FFAR. Evaluation of TGFα shedding demonstrates that propionate has EC(TIF)Click here for additional data file.S3 Fig(A) MCF7 cells were treated with vehicle control or with 100μM propionate for 24 hours, then evaluated for E-cadherin levels by western blot analysis. Results are representative of 3 independent experiments. (B) Quantitation of the relative density of the data from (A). No significant change in E-cadherin was observed (N = 3).(TIF)Click here for additional data file.S4 FigMesenchymal-like MDA-MB-436 cells and epithelial-like BT-474 cells were transiently transfected with pcDNA3.1 (vector) or FFAR2-expressing plasmids. After 48 hours, cells were treated with vehicle or 1 mM propionate. 24 hours after treatment, cell lysates were collected and evaluated for E-cadherin and β-actin proteins by western analysis. Increased E-cadherin expression was observed in MDA-MB-436 cells, but not BT-474 cells. Images are representative of 2 independent experiments.(TIF)Click here for additional data file.


The purpose of this work is to develop a highly accelerated MR technique for coronary plaque characterization with 1) whole-heart coverage, 2) fine isotropic spatial resolution, and 3) simultaneously acquired bright-blood anatomical reference.The detection of high-risk coronary atherosclerotic lesions before severe plaque complications is the "holy grail" in cardiology. Recently T1-weighted (T1w) MRI with [2] or without [3] contrast enhancement (CE) has been used for characterizing coronary plaques showing promising prognostic value [4]. However the drawbacks of current protocols based on conventional Cartesian acquisition and navigator gating hinder the clinical application of this technique: a) coverage is limited to proximal coronary segments; b) spatial resolution is low and often anisotropic; c) because normal tissue in T1w images is highly suppressed, a separate MRA acquisition is needed to provide anatomical reference. 3 mm3; spatial resolution = 1.13 mm3; TR/TE = 4.6/2.3 ms; number of radial projections = 8500; scan time = ~10 minutes depending on heart rate. After completing MRI, 21 CAD patients further underwent interventional X-ray angiography (XA) and intracoronary optical coherence tomography (OCT) for coronary plaque evaluation. OCT images were graded for high-risk coronary plaque features  by two experienced cardiologists without the knowledge of MR results.CATCH consists of ECG-gated, inversion recovery (IR) prepared spoiled gradient echo sequence with golden angle 3D radial trajectory to acquire dark-blood T1w images and bright-blood reference images in an interleaved fashion  in either pre-CE or post-CE T1w MRI. In total 3 patients showed CHIPs on pre-CE exams and 4 patients showed CHIPs on post-CE exams, respectively. Fig. The proposed MR technique of accelerated T1w whole heart coronary plaque characterization with simultaneously acquired anatomical reference was feasible. Coronary plaque hyper-intensity showed positive association with certain high-risk plaque features on OCT.


Scientific Reports 10.1038/s41598-017-15995-1, published online 17 November 2017Correction to: The competing financial interests statement in this Article should read:“The authors hold patents on this technology.”


Drosophila adult testis which has two adult stem cell lineages, the germline stem cell (GSC) lineage and the cyst stem cell (CySC) lineage, to study how chromatin factors regulate stem cell differentiation. We find that the chromatin factor Enhancer of Polycomb [E(Pc)] acts in the CySC lineage to negatively control transcription of genes associated with multiple signaling pathways, including JAK-STAT and EGF, to promote cellular differentiation in the CySC lineage. E(Pc) also has a non-cell-autonomous role in regulating GSC lineage differentiation. When E(Pc) is specifically inactivated in the CySC lineage, defects occur in both germ cell differentiation and maintenance of germline identity. Furthermore, compromising Tip60 histone acetyltransferase activity in the CySC lineage recapitulates loss-of-function phenotypes of E(Pc), suggesting that Tip60 and E(Pc) act together, consistent with published biochemical data. In summary, our results demonstrate that E(Pc) plays a central role in coordinating differentiation between the two adult stem cell lineages in Drosophila testes.Stem cells reside in a particular microenvironment known as a niche. The interaction between extrinsic cues originating from the niche and intrinsic factors in stem cells determines their identity and activity. Maintenance of stem cell identity and stem cell self-renewal are known to be controlled by chromatin factors. Herein, we use the  Drosophila adult testis as a model system, in which two types of stem cells co-exist and interact: germline stem cells and somatic stem cells. We find that a chromatin regulator called Enhancer of Polycomb [E(Pc)] acts in somatic cells to promote germ cell differentiation and maintain germ cell fate. This regulation is mediated by several signaling pathways, such as EGF and JAK-STAT pathways. E(Pc) also works with another chromatin regulator, the histone acetyltransferase Tip60, in somatic cells. Insufficient activity of the E(Pc) homolog in human leads to cancers. Our studies of E(Pc) may help understanding its roles as a tumor suppressor.Tissue maintenance and repair rely on adult stem cells, which can divide to generate new stem cells as well as cells committed for becoming specific cell types. Stem cell activity needs to be tightly controlled because insufficient or unlimited stem cell division may lead to tissue degeneration or tumorigenesis. This control depends not only on stem cells themselves, but also on the microenvironment where stem cells reside. The chromatin structure of stem cells is crucial to determine their activities. The signaling pathways connecting stem cells with their microenvironment is also important. Here we ask how chromatin factors interact with signaling pathways in determining stem cell activity. We use  In physiological condition, adult stem cells are surrounded by other cell types and extracellular matrix. Recent studies have demonstrated a dynamic crosstalk between stem cells and their surrounding microenvironment termed as the stem cell niche . SignaliDrosophila adult testis: the germline stem cell (GSC) lineage and the cyst stem cell (CySC) lineage . Forhe niche ,15,23,24cription . Moreovecription . Howevernucleosome acetyltransferase of H4) histone acetyltransferase (HAT) complex [in vivo functions of E(Pc) have been elusive.The enhancer of Polycomb [E(Pc)] gene is known as a putative Polycomb group (PcG) gene which is conserved from yeast to mammals, suggesting its crucial roles in regulating chromatin structure across species. The yeast homolog of E(Pc) was identified as a component of the NuA4  in endogenous adult stem cell lineages. We find that E(Pc) promotes cyst cell differentiation by downregulating CySC-enriched transcription factors Zfh-1 and Yan. Loss of such repression by E(Pc) in CySC lineage blocks both cyst and germ cell differentiation, which causes both CySC-like and early-stage germline tumors, including GSC-like tumor and spermatogonial tumor. In addition, when E(Pc) is specifically knocked down in CySC lineage, some germ cells ectopically turn on expression of cyst cell markers such as Zfh-1 and Yan. When ChIP followed by high-throughput sequencing (ChIP-seq) is performed specifically in cyst cells, many components of key signaling pathways are identified as direct targets of E(Pc), suggesting its central role in coordinating the crosstalk between CySC and GSC lineages. Finally, inactivation of Tip60 HAT activity in the CySC lineage leads to defects resembling loss-of-function phenotypes of E(Pc), suggesting that they act together in vivo. Thus, E(Pc) establishes proper chromatin state in cyst cells to provide an instructive microenvironment to guide germ cell differentiation and protect germline identity.Here we use the in vivo functions of E(Pc) in the Drosophila adult testis, we first characterized the E(Pc) expression pattern. Using a GFP-tagged genomic rescue transgene , the nuclear E(Pc) gDNA-GFP signal was detected in both germ cells and cyst cells throughout the adult testis (To understand the t testis . The nucBecause E(Pc) is required for early development and the null allele is lethal at embryonic or early larval stage , we firsTj>E(Pc) dsRNA (N = 32) and Tj>E(Pc) shRNA (N = 35) testes (Tj>E(Pc) dsRNA (N = 18) and 74% of Tj>E(Pc) shRNA (N = 35) testes, (Tj>E(Pc) shRNA testes (N = 31) , a transcription repressor with multiple zinc finger domains and a homeodomain. It is highly expressed in CySCs and early cyst cells, and it is required for CySC maintenance . The sec) testes , most li) testes . It has ) testes . By way  testes, . Further(N = 31) . These dE(Pc) in the entire CySC lineage, the hs-FLP; Actin-FRT-stop-FRT-Gal4, UAS-GFP; UAS- E(Pc) shRNA fly strain  was used to induce E(Pc) knockdown in a subset of cells in CySC lineage. When GFP-positive cells  and Tj-Gal4>UAS-H3-GFP, UAS-E(Pc) shRNA (N = 44) testes (E(Pc) knockdown testes  knockdown experiments primarily used ) testes . Althougn testes , consistn testes , GFP sign testes , suggestE(Pc) in cyst cells using Tj-Gal4 led to excess early-stage germ cells in 43% of Tj>E(Pc) dsRNA testes (N = 40). Further reduction of E(Pc) levels, using a loss-of-function mutant E(Pc)1 , suggesting knockdown effects, we reason that the residual E(Pc) cDNA-GFP is sufficient to rescue the E(Pc) knockdown phenotypes. Therefore, this genetic background provided a unique opportunity to immunoprecipitate E(Pc)-bound chromatin in the CySC lineage using a ChIP-grade GFP antibody /+ testes (N = 150), respectively. Furthermore, studies in the Drosophila optic lobe identified E(Pc) as one JAK-STAT target positively regulated by JAK-STAT signaling  (BL29023), UAS-yan.ACT (BL5789), Vein-lacZ (BL11749), 2X STAT-GFP , Df(zfh-1) were used. Flies with the following genotypes: Tj-Gal4/ Egfrf2; UAS-E(Pc) dsRNA/+, Tj-Gal4/+; UAS-E(Pc) dsRNA/+ were shifted to 29°C for 3D before analysis. Flies with the following genotypesTj-Gal4/yan: IP; UAS-E(Pc) shRNA/+, Tj-Gal4/+; E(z)731/ UAS-E(Pc) shRNA, Tj-Gal4/+; Df [E(z)]/UAS-E(Pc) shRNA, Tj-Gal4/+; UAS-E(Pc) shRNA/ Stat92E06346, Tj-Gal4/+; UAS-E(Pc) shRNA/zfh-175.26, Tj-Gal4/+; UAS-E(Pc) shRNA/ Df(zfh-1) were shifted to 29°C for 5D before dissection.To identify if E(Pc) cDNA-GFP in cyst cells is sufficient to rescue Tj>E(Pc) RNAi phenotype, flies with the following genotype: Tj-Gal4/ UAS-E(Pc) cDNA-GFP; UAS-E(Pc) shRNA/+, Tj-Gal4/ UAS-E(Pc) cDNA-GFP; UAS-E(Pc) dsRNA/+ were dissected at 5D after shifting from 25°C to 29°C. To test potential defects by overexpression of E(Pc) or Tip60, testes from Tj-Gal4/ UAS-E(Pc) cDNA, Tj-Gal4/ +; UAS-Tip60 cDNA/+ males 10-11D after shifting from 25°C to 29°C were analyzed.To study if expression of hs-FLP; Act5c.FRT-CD2-FRT.Gal4/+; UAS-GFP/UAS-E(Pc) shRNA and hs-FLP; Act5c.FRT-CD2-FRT.Gal4/+;UAS-GFP/+  were heat shocked at pupal stages for two days with two hours on each day. Enclosed flies were aged for 5-6D after heat shock and used for dissection and immunostaining.To analyze function of E(Pc) in individual cyst cells, flies with the following genotype: E(Pc)1 null allele was recombined with FRT42D to generate FRT42D, E(Pc)1/Cyo flies. Adult flies with following genotype: UAS-GFP hs-FLP; FRT42D, Tub-Gal80/ FRT42D, E(Pc)1; Tub-Gal4 and control flies UAS-GFP hs-FLP; FRT42D, Tub-Gal80/ FRT42D; Tub-Gal4 were aged for one day, then heat shocked for 2 hours and aged until dissection.To generate MARCM clones, UASp-E(Pc) cDNA and UASp-E(Pc) cDNA-GFP, E(Pc) cDNA was amplified using cDNA prepared from wild-type testis as the template. The 5’ half of E(Pc) cDNA was amplified as a KpnI and NotI flanked fragment with E(Pc) F1 and R1 primers. The 3’ half of E(Pc) cDNA was amplified as an NotI and XbaI flanked fragment using E(Pc) F2 and R2 primers. These two fragments were then ligated into pGEM-T-easy vector (Promega) followed by sequencing. To insert the GFP sequences at the 3’-end of E(Pc) cDNA, a Pml I site was generated right upstream of the stop codon of E(Pc) within R2 primer. GFP fragment was amplified as a Pml I and Xba I flanked fragment with Primer 5’ GFP and 3’ GFP, followed by ligation into pGEM-T-E(Pc) 3’ half cDNA opened with Pml I and XbaI restriction enzyme digestion. Finally, the 5’ half E(Pc) cDNA in a KpnI to NotI fragment and the 3’ half with and without GFP in a Not I to Xba I fragment were ligated into pBlueScript vector (Agilent Technologies) cut with Kpn I and Xba I in a 3-way ligation to generate a KpnI and XbaI flanked full-length E(Pc) cDNA tagged with GFP. Then the E(Pc) full cDNA tagged with GFP was cut with Kpn I and Xba I and ligated into UASp vector cut using same two enzymes.For transgenic fly E(Pc) genomic region was ordered from BACPAC Resources Center (BPRC). Zra I is one unique enzyme site close to the stop codon of E(Pc) genomic region. Pac I is another unique enzyme site within the 3’UTR region of E(Pc). Using primers 3’UTR F and 3’UTR R ended with Asc I and Pac I, an approximate 3 Kb fragment was amplified using the BAC clone as template and ligated into pGEM-T easy vector. Using primers GFP F and GFP R ended with Zra I and Asc I GFP sequence was amplified. Then, the GFP in a Zra I to Asc I fragment was ligated into pGEM-T 3’UTR vector, cut with AscI and PacI to generate a GFP-3’ UTR fragment flanked by ZraI and PacI. Then GFP-3’ UTR cut with ZraI and PacI was ligated into P[acman], opened with ZraI and PacI to generate E(Pc) genomic plasmid tagged with GFP.To generate E(Pc) genomic plasmid tagged with GFP, a 21 kb P[acman] BAC clone (CH322-140G22) covering the entire Transgenic fly lines were generated by Bestgene Inc . More than three independent transgenic lines were generated for each transgene.Primers:E(Pc) F1: GGGGTACCATGTCCAAGCTGTCGTTCAGAGCCCE(Pc) R1: ATAAGAATGCGGCCGCTGCCGCCGAE(Pc) F2: ATAAGAGCGGCCGCCGCTCGTGCE(Pc) R2: GCTCTAGATCACACGTGTCTGTTGATGGTTGACGTCACAC5’ GFP: ATCACGTGATGGTGAGCAAGGGCGAGGAG3’ GFP: GCTCTAGATTACTTGTACAGCTCGTCCATGCCG3’UTR F: GGCGCGCCGACGCGGATGGCAGCG3’UTR R: CCTTAATTAAACATACATACGTATTCTTTTTGTTTTGGGFP F: GACGTCAACCATCAACAGAATGGTGAGCAAGGGCGAGGFP R: GGCGCGCCTTACTTGTACAGCTCGTCCATGCDrosophila embryos, DSHB, 8B12H9), GFP , dpERK , HA , β-Galactosidase . For dpERK staining, testes were dissected in 10 mM Tris-Cl pH 6.8, 180 mM KCl, 50 mM NaF, 1 mM Na3VO4, 10 mM b-glycerophosphate as described before . The edgeR method models short reads into negative binomial distribution and estimates the biological replicate variance (dispersion). Tag-wise dispersion estimation was performed in “Tj-Gal4”, “Tj-Gal4/+; E(Pc) shRNA/+” two groupings of read count profiles. We introduced quantity term “corrected RPKM (cRPKM)” by the formula: pseudo.alt * 1e+09 / (length of merged transcripts)/ (common.lib.size). The common.lib.size was calculated from the calcNormFactors function of edgeR, which performs TMM and upper quantile normalization methods and set a reference library. The pseudo.alt contains read counts after normalization across the input multiple profiles. The pseudo.alt was calculated by edgeR using quantile normalization and maximum likelihood method. The pseudo.alt contains pseudo read counts after correcting the library size and composition differences.To identify differentially expressed genes, we utilized the edgeR software package  in R to R , was utilized to visualize the global similarity relationship of 4 samples.After cRPKM calculation, gene expression levels per sample were pair-wisely compared with spearman correlation (correlation coefficient rho). A pair-wise inter-profile distance was defined as (1-rho) and set up a distance matrix. A dimension reduction method, multidimensional scaling in GEO accession number for ChIP-seq and RNA-seq data is GSE93828.S1 FigA-A”) GFP signal in the nuclei of both germ cells (yellow arrowheads) and Eya-positive late-stage cyst cells (yellow arrows). Scale bar: 50μm. (B-B”) At the apical tip, GFP signal in the nuclei of germ cells (white arrowhead in B’ and yellow arrowhead in B”), early cyst cells  and later cyst cells . (C-C”) In Tj>E(Pc) RNAi, E(Pc) gDNA-GFP testes, undetectable GFP signal in early cyst cells  and later cyst cells , but detectable in germ cells (white arrowhead in C’ and yellow arrowhead in C”). Asterisk: hub. Scale bar: 20μm.((TIF)Click here for additional data file.S2 FigA) Percentage of testes with <10, 10–30 and >30 Zfh-1- and Eya-double positive cyst cells in different genotyped testes. (B-C) Immunostaining with anti-Tj and Eya in Tj-Gal4 and Tj>E(Pc) shRNA testes. (D) Quantification of Tj-positive cells in Tj-Gal4 control testes: 50 ± 12.49  and in Tj>E(Pc) shRNA testes: 83.91 ± 22.41 (N = 31). Quantification of Eya-positive cells at the tip of Tj-Gal4 control testes: 39 ± 7.35  and Tj>E(Pc) shRNA testes: 58 ± 13.04 (N = 43). **** P<0.0001, two-tailed t test. (E-F’”) Immunostaining using the germ cell marker Vasa  and a late cyst cell marker Eya  in Tj>H3 GFP and Tj> H3 GFP, E(Pc) shRNA testes. Asterisk: hub. Scale bar: 20μm.((TIF)Click here for additional data file.S3 FigC and D, green in A, B’, D”), early cyst cell markers Zfh-1  and Yan , hub marker Armadillo, as well as spectrosome/fusome marker α spectrin  in Tj>E(Pc) shRNA testes. (B-B’) Over-proliferating germ cells within one cyst  had both round spectrosome (yellow arrowhead) and branched fusome (yellow arrow). Scale bar: 20μm.Immunostaining using the germ cell marker Vasa ((TIF)Click here for additional data file.S4 FigA-A’) In Bam-HA, Tj-Gal4 control testes, immunostaining with anti-HA (red) and anti-Vasa (green) showed Bam expression in 4- to 16- spermatogonial cells (red dashed line). In Bam-HA, Tj>E(Pc) dsRNA testes (B-B’) and Bam-HA, Tj>E(Pc) shRNA testes (C-C’): Bam was detectable in spermatogonial tumor cells . Asterisk: hub. Scale bar: 20μm.((TIF)Click here for additional data file.S5 FigA-A”) In nos>upd testes, Vasa-positive GSC-like cells  were intermingled with Zfh-1-positive cells . Scale bar: 20μm. White dashed region enlarged in B-B”. Vasa-positive cells  were not stained with antibodies against Zfh-1 . Scale bar: 10μm. (C-C”) In bam1/bam114 testes, spermatogonial tumor cells (white dashed circle) were not stained with antibodies against Zfh-1. Scale bar: 50μm. (D-D”) Enlarged apical tip (white dashed square in C-C”): Zfh-1 only detectable at the apical tip (arrowhead in D-D”). Scale bar: 20μm.((TIF)Click here for additional data file.S6 FigA-C) In Tj>E(Pc) shRNA testes, E(Pc) knockdown in cyst cells led to both somatic and germline tumor shown as expansion of DAPI bright region (white dashed line). Scale bar: 100μm. (D) Quantification of the penetrance and severity of the tumor phenotype at different genetic backgrounds. Testes were dissected from flies 5 days after shifting to 29°C. **P<0.01, ****P<0.0001, chi-square test.((TIF)Click here for additional data file.S7 FigA-A’”) In upd-Gal4 control testes, transit-amplifying stage germ cells (yellow dashed line) with DAPI bright nuclei localize at the apical tip of testis. (B-B’”) In upd>E(Pc) dsRNA testes, no expansion of DAPI bright region was observed as in Tj>E(Pc) RNAi testes. Refer to ((TIF)Click here for additional data file.S8 FigA-B”’) 5D After clonal induction (ACI), GFP labeled wild-type CySCs (yellow arrowhead) were Zfh-1 positive, while GFP positive cyst cells (yellow arrows) had none (A”) or diminished Zfh-1 expression (B”). (C-C”’) 5D ACI, Zfh-1 was still detectable in GFP-labeled Eya-positive E(Pc) mutant cyst cells (yellow arrows). Asterisk: hub. Scale bar: 10μm. (D-D’) GFP positive CySCs localized at the apical tip DAPI bright region. In the same testes (E-E’), extra DAPI bright cells (yellow dashed line), including Zfh-1-positive E(Pc) mutant cyst cells (yellow arrow in D), were detected. Asterisk: hub. Scale bar: 20μm.((TIF)Click here for additional data file.S9 FigA-B) Tj>E(Pc) cDNA-GFP and Tj>E(Pc) cDNA-GFP, E(Pc) shRNA testes were mounted on the same slide for comparing the GFP signal. Asterisk: hub. Scale bar: 20μm. (C) Quantification of the GFP intensity. Tj>E(Pc) cDNA-GFP: 33141 ± 6499 ; Tj>E(Pc) cDNA-GFP, E(Pc) shRNA: 11523 ± 3811 (N = 50). **** P<0.0001. Two-tailed t test.((TIF)Click here for additional data file.S10 FigA-B) Immunostaining using Vasa (germ cell marker) and Zfh-1 (early cyst cell marker) in Tj-Gal4 control testes (A) and Tj>E(Pc) cDNA testes (B). GSCs labeled by white dots and Zfh-1 positive cells by white arrowhead. Asterisk: hub. Scale bar: 20μm. (C) Quantification of Zfh-1-positive cells. Tj-Gal4: 31.28 ± 6.69 ; Tj>E(Pc): 20.97 ± 5.62 (N = 68). (D) Quantification of GSCs. Tj-Gal4: 8.31 ± 2.04 (N = 75); Tj>E(Pc): 4.95 ± 1.35 (N = 78). *** P<0.001. Two-tailed t test. (E-F) Immunostaining using a pan cyst cell marker Tj in Tj-Gal4 control testes (E) and Tj > E(Pc) testes (F). Asterisk: hub. Scale bar: 20μm. (G) Quantification of Tj-positive cells. Tj-Gal4: 56.26 ± 9.61 ; Tj>E(Pc): 51.19 ± 9.69 (N = 47). *P<0.05. Two-tailed t test.((TIF)Click here for additional data file.S11 FigA-B) Quantification of the penetrance and severity of the germline tumor phenotype at different genetic background. ***P<0.001, ****P<0.0001, chi-square test. (C) Quantification of Zfh-1-positive cells. Tj-Gal4 control testes: 40±6.96 (N = 35), Tj>Tip60 cDNA testes: 30.42±8.24 (N = 50). ****P<0.0001, Two-tailed t test. (D) Quantification of GSCs. Tj-Gal4 control testes: 8.11±1.84 (N = 35), Tj>Tip60 cDNA testes: 6.64±2.18 (N = 50). **P<0.01, Two-tailed t test.((TIF)Click here for additional data file.S12 FigTj-Gal4, Tj >E(Pc) shRNA testes for tetra-acetylated histone H4 (H4 ace) were analyzed by qPCR. Enrichment of H4 ace at zfh-1 (A) and yan (B) loci were normalized to input as percentage of input. Error bars, s.d. for N = 2 biological replicates. P value: two-tailed t test.ChIPed DNA in (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


FOXP1 gene, inhibits FOXP1 induction of TAM differentiation, and the secretion of inflammatory cytokines into the tumor microenvironment, inducing the proliferation of NPC and GC cells. FOXP1 overexpression hindered monocyte differentiation and inhibited NPC and GC cells growth. Our results demonstrated that EBV-miR-BART11 plays a crucial role in the promotion of inflammation-induced NPC and GC carcinogenesis by inhibiting FOXP1 tumor-suppressive effects. We showed a novel EBV-dependent mechanism that may induce the carcinogenesis of NPC and GC, which may help define new potential biomarkers and targets for NPC and GC diagnosis and treatment.Epstein-Barr virus (EBV) infection and chronic inflammation are closely associated with the development and progression of nasopharyngeal carcinoma (NPC) and gastric cancer (GC), and the infiltration of inflammatory cells, including tumor-associated macrophages (TAMs), is often observed in these cancers. EBV encodes 44 mature micro RNAs (miRNAs), but the roles of only a few EBV-encoded miRNA targets are known in cancer development, and here, our aim was to elucidate the effects of EBV-miR-BART11 on FOXP1 expression, and potential involvement in inflammation-induced carcinogenesis. We constructed an EBV miRNA-dependent gene regulatory network and predicted that EBV-miR-BART11 is able to target forkhead box P1 (FOXP1), a key molecule involved in monocyte to macrophage differentiation. Here, using luciferase reporter assay, we confirmed that EBV-miR-BART11 directly targets the 3′-untranslated region of  Chronic inflammation is an important mediator of nasopharyngeal carcinoma (NPC) –3 and gaEpstein-Barr virus (EBV) infection is closely associated with the development and progression of NPC and GC –13. EBV FOXP1) mRNA. FOXP1 has been reported to be involved in monocyte differentiation to macrophage, where its downregulation represents a key molecular event [We previously profiled all 44 EBV-encoded mature miRNAs in NPC biopsies and non-cancerous nasopharyngeal tissues, and found that EBV miRNAs located in the BART region were highly expressed in NPC biopsies –33. Addiar event , 35. HowFOXP1 3′-untranslated region (UTR)  in the ) Figure . To dete) Figure . These r) Figure .FOXP1 3′-UTR were co-transfected with the EBV-miR-BART11 precursor expression vector in 5-8F cells. EBV-miR-BART11 significantly attenuated the luciferase activity of FOXP1-WT vectors II and III, but exhibited no effects on the FOXP1-WT-I vector or the FOXP1-mutant vectors  EBV-miR-BART11 binding or mutant sequences of the s Figure . These rFOXP1 mRNA expression was assessed in 30 NPC biopsies and 10 non-tumor nasopharyngeal epithelial tissues. As expected, EBV-miR-BART11-3p and EBV-miR-BART11-5p expression levels were significantly higher in NPC samples than in normal nasopharyngeal epithelial samples, and these levels were negatively correlated with FOXP1 expression  and In order to define the relationship between EBV-miR-BART11 and FOXP1 in monocyte to macrophage differentiation further, we monitored temporal FOXP1 expression in THP-1 monocytes subjected to PMA-induced macrophage differentiation. This revealed that FOXP1 is dramatically downregulated during monocyte to macrophage transformation Figure , which iMacrophages play a key role in chronic inflammation and can trigger a pro-inflammatory response by secreting inflammatory factors , 37. TheTAMs are a major component of the immune infiltrates present in the NPC microenvironment, where they produce cytokines, growth factors, and angiogenic inducers that amplify the inflammatory response and promote tumor survival and growth . The resCytokines stimulated by the local inflammatory response promote epithelial cell carcinogenesis through the induction of cell proliferation , 9. In oOur previous study indicated that LPS-stimulated TAMs can promote epithelial cell proliferation . In ordeNuclear factor κB (NF-κB) orchestrates the inflammatory response by regulating the expression of numerous genes , 36. TheChronic, non-resolving inflammation plays an important role in the initiation of a variety of tumors, including NPC , 39 and FOXP1 locus is located on 3p14.1 [EBV-infected macrophages induce an inflammatory response in EBV-related human tumors –46, but n 3p14.1 , a region 3p14.1 –54, and n 3p14.1 , 56 in Nn 3p14.1  and T lyn 3p14.1 , monocytn 3p14.1 , 59, andFOXP1 as a target of EBV-miR-BART11 in both monocytes and epithelial cells, and showed that FOXP1 downregulation was necessary for monocyte differentiation. We further investigated the effects of EBV-miR-BART11 and FOXP1 on macrophage cytokine secretion. EBV-miR-BART11 overexpression in TAMs resulted in LPS hyperresponsiveness and the increased secretion of cytokines, chemokines, and growth factors that support tumor survival, proliferation, and invasion. Taken together, our results indicate that EBV-miR-BART11 is sufficient to maintain and potentiate the inflammatory environment by enhancing macrophage differentiation and responsiveness through the downregulation of FOXP1.We confirmed Under certain conditions, tumor cells can induce their own inflammatory factor secretion, in order to intensify the local inflammatory microenvironment, resulting in further TAM and lymphocyte infiltration . TherefoFOXP1 acts as a transcriptional repressor in lymphocytes where its downregulation plays important roles in immune cell differentiation. It is also frequently downregulated in solid tumors, including breast cancer –63, non-NF-κB transcriptional regulation plays a critical role in tumorigenic inflammation. FOXP1 and NF-κB interact through a complex regulatory mechanism involved in several biological processes that vary in a cell type-dependent manner. In lymphocytes, FOXP1 is able to inhibit directly apoptotic gene transcription and cooperate with NF-κB, promoting human B cell survival, while FOXP1 expression is dependent on NF-κB transcriptional activity and plays a synergistic role in NF-κB self-activation . NF-κB aIn conclusion, we revealed EBV-miR-BART11 plays an important role in the inflammatory microenvironment and inflammation-induced carcinogenesis in EBV-associated cancers through the direct inhibition of FOXP1 and NF-κB activation. Collectively, these data demonstrate that EBV-miR-BART11 or FOXP1 may serve as potential diagnostic or prognostic markers in NPC or GC, and may represent important targets for EBV-related cancer immunotherapy.2. THP-1 cells were treated with 10 nM phorbol 12-myristate 13-acetate  to induce monocyte differentiation. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 was obtained from Sigma (10 ng/mL). 293T cells used to produce the lentiviral stock were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, at 37°C in the atmosphere containing 5% CO2.Two EBV-negative human NPC cell lines (5-8F and HK-1), AGS GC cells, and THP-1 monocytes were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) at 37°C in the atmosphere with 5% COFOXP1 full-length coding sequence  was amplified and cloned into the pIRESneo3 vector (Invitrogen). Cell transfection was performed in 70-80% confluent cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol.FOXP1-specific siRNAs and their corresponding controls were synthesized by Ruibo  . EBV-miRFOXP1 CDS was subcloned into the pLenti6/V5-D-TOPO vector. pLenti6/V5-D-TOPO/BART11 or pLenti6/V5-D-TOPO/FOXP1 vectors and ViraPower Packaging Mix were co-transfected using a gene carrier kit  into 293T cells to produce a lentiviral stock, and 48 h after transfection, virus-containing supernatant was collected and used to infect THP-1 at a ratio of 1:1 with fresh medium, as described previously [Lentiviral vector construction was constructed using the ViraPower™ Lentiviral Expression System (Invitrogen). First, EBV-miR-BART11 precursor or full-length eviously .β-actin or GAPDH as the internal controls. The primers used are shown in Total RNA was harvested using the TRIzol Extraction Kit (Invitrogen) and cDNA samples were prepared using a QuantiTect Reverse Transcription Kit . Stem-loop real-time qRT-PCR for mature miRNAs was done with the Qiagen QuantiTect SYBR Green PCR Kits (Qiagen), using RNU6B (U6) as an international control. qRT-PCR for mRNA expression was performed using the SYBR Premix Ex Taq II kit  according to the manufacturer instructions with Whole cell lysates were extracted using RIPA lysis buffer . Protein concentration was quantified using a BCA protein assay kit . Afterward, 50 μg of protein was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a PVDF membrane . Immunodetection using antibodies against FOXP1 , NF-κB p65 , or β-actin (Millipore) was done for 1 h at room temperature or overnight at 4°C. Antibody-antigen complexes were detected using the enhanced chemiluminescence (ECL) system . β-actin served as a loading control.Cell proliferation was measured using an MTT (Millipore) assay. After transfection or treatment, 800 cells per well were seeded in 96-well plates, and viability was assessed in five replicates at designed times. The formation of colored formazan dye was measured at 490 nm on a spectrophotometric plate reader.For the clonogenic assays, 1000 cells were plated in six-well plates at 24 h post-transfection and incubated for 14 days. The number of colonies was counted with Image-Pro Plus  and normalized to the control group. The experiments were performed at least three times.Cell cycle analysis was performed using flow cytometry with propidium iodide (PI) staining (5 μg/mL) 48 h post-transfection. Events were recorded from samples using a FACSCalibur Flow Cytometer  equipped with Cell Quest 3.3 software and analyzed using ModFit software .Renilla luciferase vector . For NF-κB reporter assay, 5-8F cells were transiently co-transfected with the pNF-κB-Luc construct (Promega), pRL-TK vector, and EBV-miR-BART11 or the FOXP1 expression vector or siFOXP1. Luciferase activity was determined by the Dual Luciferase Assay Kit (Promega) 48 h after transfection [5-8F cells were co-transfected with synthetic EBV-miR-BART11 and luciferase reporter vectors (FOXP1-WT or FOXP1-mutant) along with pRL-TK sfection –81. TranFOXP1 gene expression was analyzed using Spearman's correlation coefficient. p < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 13.0  and Graph Pad Prism 5 . Data are presented as mean ± standard deviation (SD). Comparisons between two groups were performed using Student's t-test or one-way ANOVA, unless otherwise indicated. The association between EBV-miR-BART11-(3p and 5p) and


Tamoxifen effectively induced CGRPcre-ER production in DRG. ≈87% of CGRPcre-ER-expressing neurons were co-labeled CGRP antibody. Three small and two medium-large-sized (5HT3a+/NPY2R- and NPY2R+) neuronal groups with unique electrophysiological profiles were CGRP-cre+. Nav1.8-cre+ neurons were detected in all CGRP-cre+ groups, as well as in 5 additional neuronal groups: MrgprD+/TRPA1-, MrgprD+/TRPA1+, TRPV1+/CGRP-, vGLUT3+ and ≈30% of trkC+ neurons. Differences between TRPV1cre and Nav1.8cre reporters were that unlike TRPV1-cre+, Nav1.8-cre+ expression was detected in non-nociceptive vGLUT3+ and trkC+ populations. Many TRPV1-cre+ neurons did not respond to capsaicin. In contrast, V1-GFP+ neurons were in 4 groups, each of which was capsaicin-sensitive. Finally, none of the analyzed reporter lines showed cre-recombination in trkB+, calbindin+, 70% of trkC+ or parvalbumin+ neurons, which together encompassed ≈20% of Nav1.8-cre- DRG neurons. The data presented here increases our knowledge of peptidergic sensory neuron characteristics, while showing the efficiency and specificity manipulation of peptidergic neurons by the CGRPcre-ER reporter. We also demonstrate that manipulation of all C- and A-nociceptors is better achieved with TRPV1-cre reporter. Finally, the described approach for detailed characterization of sensory neuronal groups can be applied to a variety of reporter mice.Peptidergic sensory neurons play a critical role in nociceptive pathways. To precisely define the function and plasticity of sensory neurons in detail, new tools such as transgenic mouse models are needed. We employed electrophysiology and immunohistochemistry to characterize in detail dorsal root ganglion (DRG) neurons expressing an inducible CGRP It is now recognized that somatosensory neurons are neurochemically, functionally and physiologically diverse ,2,3. Thi+) ;,22;+, Naof +20mV  [21]; anT (30μM)  or mustaT (30μM)  to evalum in pF) values.Cells were considered spherical, and therefore, diameter (d in μm) of cells was calculated from capacitance (Cm to AP peak), fall time  and duration at the base  as well as 80% recovery time of after-hyper-polarization to baseline (AHP80) were measured from data generated by protocol-1  exponential function.AP rise time  calculation was performed using pCLAMP10.2 software . Presenc2+ imaging as previously described [2+ ([Ca+2]i) was collected for 60 s prior to agonist applications. The net changes in Ca+2 influx were calculated by subtracting the basal level of [Ca+2]i from the peak [Ca+2]i value achieved after exposure to the agonists. Ca2+ imaging experiments were combined with patch-clamp recording.MO responses were acquired with fluorescent Caescribed . Data wecre-ER/-;Rosa26LSL-tDTomato/- (i.e. double heterozygotes) and Nav1.8cre/-;Rosa26LSL-tDTomato/- 4% paraformaldehyde-perfused mice. Tissues were additionally fixed with 4% paraformaldehyde for 15 min, cryo-protected overnight with 30% sucrose in phosphate buffer, embedded in Neg 50 ; and 30μm cryo-sections were generated as previously described . S7 lication . This wo Aβ-HTMR  5,22]. . +, Nav1R family  9]. M4 . M4 +, Noceptors  46]. Si. Si+, Nacre-recombination in the CGRPcre-ER/+ reporter mice occurs in 84% of peptidergic (i.e. CGRP-ab+) neurons. Possible leak into non-peptidergic nociceptors or LTMR-classified neurons is minimal if any  are likely belonging to PEP-1, PEP-2 and NP-2 clusters  as well as some LTMRs (S7 and M4 groups)  ; Fig 11 tors S4-S groups a3 groups  [1]. Neuiceptors ; Fig 11.cre-ER/+ and TRPV1cre/+ reporter mice in the manipulation of sensory neurons, TRPV1 and to lesser extent CGRP expression could be detected outside sensory neurons. Thus, TRPV1 is expressed at low-to-medium levels in the immune system [Despite dominat expression of CGRP and TRPV1 in sensory neurons, and many advantages of CGRPe system , epithele system  and othee system . Outsidee system ,62. Thuse system , and thee system . Additioe system .In vivo intracellular recording from DRG and TG neurons gives the utmost precision in determining sensory neuronal types [ex-vivo tissue-nerve preparations also produce reliable results [ex vivo electrophysiology approaches combined with ontogenetic are gaining popularity [in vivo intracellular recording makes clear the link between AP/AHP and the corresponding function of sensory neuronal groups disregarding whether neurons innervate paw, vibrissa pad or muscle [Identification and determination of sensory neuron functional groups were carried out by several approaches. al types ,42, espeal types . Extrace results . Howeverpularity . Accumulr muscle ,41,42. Sr muscle  (especiar muscle ,12 and dr muscle ,56,58,65cre-ER/+ is an effective mouse reporter line for manipulation of C- and A-peptidergic nociceptors. Our data also indicates that targeting nociceptors, but not LTMR neurons can be optimally achieved in TRPV1cre/+ reporter mouse lines, while some LTMR neurons could potentially be affected in Nav1.8cre/+ mice. Finally, presented here sensory neuron group identification approach could successfully be used for numerous other reporter mouse lines.In conclusion, our data show that CGRP


The impact of metabolic stress induced by obesity on the bone marrow melanoma niche is largely unknown. Here we employed diet induced obese mice model, where mice received high-fat (HFD) or normal diet (ND) for 6 weeks before challenge with B16F10 melanoma cells. Tumor size, bone loss and osteoclasts numbers were assessed histologically in the tibial bones. For defining the molecular pathway, osteopontin knock-out mice, interleukin 6 neutralizing antibody or Janus kinase 2 inhibition were carried out in the same model. Mechanistic studies such as adipocyte-melanoma co-cultures for defining adipocyte induced changes of tumor cell proliferation and expression profiles were also performed. As results, HFD enhanced melanoma burden in bone by increasing tumor area and osteoclast numbers. This process was associated with higher numbers of bone marrow adipocytes expressing IL-6 in direct vicinity to tumor cells. Inhibition of IL-6 or of downstream JAK2 blocked HFD-induced tumor progression. Furthermore, the phenotypic changes of melanoma cells triggered macrophage and osteoclast accumulation accompanied by increased osteopontin expression. Osteopontin triggered osteoclastogenesis and also exerted a positive feedback loop to tumor cells, which was abrogated in its absence. Metabolic stress by HFD promotes melanoma growth in the bone marrow by an increase in bone marrow adipocytes and IL-6-JAK2-osteopontin mediated activation of tumor cells and osteoclast differentiation. Around 39% of the world adult population is overweight, and 13% obese. Epidemiological, clinical and experimental studies have shown that over nutrition leading to adipose tissue expansion increases the risk of malignant melanoma (MM) and worsens its prognosis . Bone meThe bone marrow hematopoietic stem cell (HSC) niche is key for cancer cells to engraft into bone –18. DuriAdipocytes, however, are not only present in subcutaneous fat tissue but also found in large quantities in the bone marrow (BM), where mesenchymal stem cells differentiate into the bone forming osteoblasts or into adipocytes. Balance between bone marrow osteoblasts and adipocytes assures the maintenance of the bone mass and the integrity of the bone marrow space. Indeed, BM adipocyte content increase with obesity . As boneIn the present study, we defined the role of obesity on the homing and survival of melanoma cells in the bone marrow niches. We demonstrate that diet-induced obesity fosters the activation of tumor-associated macrophages and osteoclasts by bone marrow adipocytes via IL-6. We further delineate that expression of osteopontin, IL-6 and the melanoma derived chemokine C-X-C motif ligand 1 (CXCL1) in the bone marrow increases after high fat diet, predisposing melanoma cells to home into the bone likely due to enhanced osteoclast activation. Most convincingly, blockade of IL-6 or osteopontin rescued obesity-induced melanoma growth into the bone marrow and normalized osteoclast activation.Cyclin D1 mRNA levels in tumor cells of HFD compared to ND mice -induced obesity impacts melanoma growth in bone, 6 week-old C57BL/6N male mice were fed for 6 weeks with HFD  or normal diet . Next, HFD and ND-treated mice were injected with B16F10 melanoma cells into the tibial bone marrow Figure . In the  Figures .cathepsin K, c-Fos, matrix metalloproteinase 9 (MMP-9), intercellular adhesion molecule 1 (ICAM1), transforming growth factor β1 (TGFβ1), connective tissue growth factor (CTGF), chemokine (C-X-C motif) receptor 2 (CXCR2), CCAAT/enhancer-binding protein beta (CEBPβ), notch homolog 1, translocation-associated (Notch1) and vascular cell adhesion molecule 1 (VCAM1) in HFD- compared to ND-treated mice 7 days after tumor cell challenge , v-akt murine thymoma viral oncogene homologue 1 (Akt1), mitogen-activated protein kinase 3 (MapK), forkhead box a1 (Foxa1), ICAM1, cadherin 2 (N-cad), C-X-C chemokine receptor type 7 (CXCR7), vascular endothelial growth factor-C (VEGF-C), transforming growth factor-β2 (TGFβ2) and angiopoietin-2 (Ang2) in tumor cells treated with HFD-derived serum (in vitro and in vivo.To determine whether circulating factors present in high fat diet (HFD) mice could influence melanoma cell proliferation ed serum , while nNext, we tested whether melanoma cells exposed to HFD serum affect osteoclastogenesis. Indeed, quantification of TRAP+ cells resembling bone-resorbing osteoclasts showed that melanoma cells exposed to HFD-serum significantly enhanced osteoclast differentiation Figures . HoweverSince obesity is known to induce inflammation , 34, we int cells in HFD compared to ND mice  mice and neutralizing IL-6 monoclonal antibody treatment, respectively. Interestingly, OPN-/− mice showed significantly reduced tumor burden and significantly lower osteoclast numbers, when challenged with HFD, compared to wild-type controls Figures , suggestint cells and systemic IL-6 levels were abolished . Mice were fed with normal diet  or high fat diet  ad libitum for 6 weeks. Intratibial injections of B16F10 cells were performed as previously described by Bakewell et al. [4 B16F10 melanoma cells suspended in 50μl PBS or only PBS (vehicle) were injected into the tibiae of anaesthetized mice. The 27G¾ needle  was inserted into the mouse tibia for delivering the cells into the metaphysis. For blocking IL-6, 0.5 mg of IL-6 monoclonal antibody  was injected intraperitoneally (i.p.) every other day after the injection of B16F10 cells into the tibia  were purchased from Jackson Labs and maintained at 25°C with 12-h light and dark cycles. OPN deficient mice were purchased from Jackson Labs (B6.128S6(Cg)-Spp1l et al. . Brieflya Figure . For Jana Figure . All mic2, 95% air). B16F10-cells were starved in serum-free α-MEM medium containing 1% antibiotics and antimycotics for 48 h. Then, 5×105 cells/well or 5×104 cells/well were plated into 6-well plate or 12-well plate, respectively and stimulated with medium containing 2% serum from normal diet (ND) or high fat diet (HFD) treated mice. For adipocyte/B16F10 co-cultures, bone marrow mesenchymal stem cells were prepared as previously described by Soleimani and colleagues [Murine melanoma cell lines (B16F10) were obtained from the American Type Culture Collection (ATCC), and were maintained in alpha minimum essential medium  containing 100 ml/L of fetal bovine serum  with 100 IU/ml penicillin and 10 μg/ml streptomycin  at 37°C  or high fat diet (HFD) mice. Pictures were taken with phase contrast microscope and 10 × magnifications for multiple views of the scratched monolayer. Coverage rate was calculated as wound width covered (%) by comparing with initial scratch size (0 h) using ImageJ software .Mouse femurs and tibias were fixed in 4% formalin and decalcified in 14% EDTA until the bone is flexible. Long bones were embedded in paraffin and sliced at equivalent coronal sections through the center of the bone with 1-2 μm thickness. Histological sections were stained with hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) activity. For the detection of TRAP positive cells in bones, 2 μm sections were proceed according the manufacturer protocol (Sigma 387A). Trabecular bone area was measured according to standard protocol  with theDe-paraffinized, ethanol-rehydrated tissue sections were stained with monoclonal antibodies (mAb) against surface markers of melanoma , IL-6 , OPN , and Ki-67 . Pre-treatment and revelation procedure were performed following the instruction of VECTASTAIN ABC Kit . For fluorescence immunostaining, slides were treated with 0.2% Triton X-100 and blocked with 5% normal serum control after antigen retrieval with citrate buffer , and incubated with the primary antibody and then secondary antibodies. Slides were covered using a DAPI mounting medium (Vector Laboratories Inc. Cat.#H-1200). Images were acquired using a Nikon Eclipse 80i microscope, equipped with Sony DXC-390P digital camera and NIS-Elements BR2.2 software.TM reagent . mRNA was isolated according to the manufacturer's instructions, digested with DNaseI and reversely transcribed into cDNA using High-Capacity RNA-to-cDNA™ Kit . Quantitative real-time PCR was performed using SYBR Green I-dTTP  on CFX96 Touch™ Real-Time PCR Detection System . Samples were analyzed in triplicates. HPRT and β-actin were used as housekeeping genes. Primer sequences are listed in Table Frozen long bones or cell lysates were homogenized with a Precellys  in peqGOLD RNAPure2, 95% air). 5×105/well (24-well plate) of non-adherent cells were cultured in α-MEM supplemented with 5 ng/ml receptor activator of nuclear factor (NF)-κB ligand (RANKL)  and 20 ng/ml M-CSF . Medium was replaced every 3 days. Cells were cultured at 37°C for 5-7 days. Mature osteoclasts were either stained for TRAP, a marker of osteoclasts, using a Sigma TRAP kit  or harvested for RNA isolation. TRAP+ multi-nucleated (n ≥ 3) cells were considered as osteoclasts. The procedure for tumour cell/osteoclast co-culture was adapted from Yagiz and colleagues [4 B16F10 cells were pre-incubated with 2 % serum from ND or HFD mice at 37°C for 12h. The cells were then fixed with 2.5% glutaraldehyde for 1 min and immediately quenched with 1.5% glycine in PBS for 20 minutes. Fixed cells were washed and incubated overnight in complete medium before the co-culture with monocytes. The cells were cultured in α-MEM medium containing 5 ng/ml RANKL and 20 ng/ml M-CSF. Medium was replaced every 3 days. TRAP staining and TRAP+ quantification were performed.For primary osteoclast assays, bone marrow (BM) cells from C57BL/6 mice (6-8 weeks) were incubated at 37°C in (100mm dish) in basal culture medium containing 5 ng/ml macrophage colony-stimulating factor (M-CSF) overnight (5% COlleagues . BrieflyBone marrow (BM) cells were isolated by flushing long bones with PBS and filtering through a 70-μm cell strainer . After red blood cell lysis, BM cells were analysed using PE-conjugated CD11b , FITC-conjugated Gr-1  and APC-conjugated Ly6C  antibodies with Gallios flow cytometer .Chemokines and cytokines were measured using Mouse Th1/Th2 10plex FlowCytomix Multiplex kit  according to the manufacturer's instruction. Serum levels of IL-6 , osteopontin , leptin , and CXCL-1  were measured using DuoSet ELISA Development kits according to the manufactured recommendations.Electrophoresis of 15 mg protein was performed. Following protein transfer into 0.45 μM nitrocellulose (NC) membrane , membranes were blocked with 5% skim-milk in TBS-T buffer (0.05%) for 2 h. Then, membranes that washed and were probed using the following antibodies: Nuclear factor kappa B (NF-κB) p65 , phospho-NF-κB p65 (Ser468)  and anti–β-actin mouse mAb , and the secondary antibodies. Chemiluminescent reagent  and detected on Amersham Hyperfilm ECL  were used for the revelation of the western blot.t test or one-way ANOVA using GraphPad Prism software 5.0 .All data are presented as mean ± SEM. The statistical significance was determined by student's


Arabidopsis pathogen-responsive mitogen-activated protein kinases (MPKs), is essential to ETI mediated by both coiled coil-nucleotide binding site-leucine rich repeats (CNLs) and toll/interleukin-1 receptor nucleotide binding site-leucine rich repeats (TNLs) types of R proteins. MPK3/MPK6 activation rapidly alters the expression of photosynthesis-related genes and inhibits photosynthesis, which promotes the accumulation of superoxide (2O2), two major reactive oxygen species (ROS), in chloroplasts under light. In the chemical-genetically rescued mpk3 mpk6 double mutants, ETI-induced photosynthetic inhibition and chloroplastic ROS accumulation are compromised, which correlates with delayed hypersensitive response (HR) cell death and compromised resistance. Furthermore, protection of chloroplasts by expressing a plastid-targeted cyanobacterial flavodoxin (pFLD) delays photosynthetic inhibition and compromises ETI. Collectively, this study highlights a critical role of MPK3/MPK6 in manipulating plant photosynthetic activities to promote ROS accumulation in chloroplasts and HR cell death, which contributes to the robustness of ETI. Furthermore, the dual functionality of MPK3/MPK6 cascade in promoting defense and inhibiting photosynthesis potentially allow it to orchestrate the trade-off between plant growth and defense in plant immunity.Extensive research revealed tremendous details about how plants sense pathogen effectors during effector-triggered immunity (ETI). However, less is known about downstream signaling events. In this report, we demonstrate that prolonged activation of MPK3 and MPK6, two  Arabidopsis plants and show that prolonged activation of MPK3 and MPK6, two kinases critical for pathogen resistance, results in the inhibition of photosynthesis and accumulation of reactive oxygen species (ROS) in the chloroplasts. We find that this response is similar to that observed during pathogen effector-triggered immunity (ETI). Correspondingly, plants that carry mutant versions of MPK3 and MPK6 result in compromised ETI-induced photosynthetic inhibition and chloroplastic ROS accumulation; thus, these two kinases seem to be essential for ETI. Our results suggest that MPK3/MPK6 activation induces a global down-regulation of photosynthesis along with an up-regulation of defense-related genes, and coordinates the growth and defense trade-off in plants.Plants follow different strategies to defend themselves against pathogens and activate their immune systems once the pathogens have been detected. One of the responses observed is the inhibition of photosynthesis and the global down-regulation of genes that regulate this process, similar to what is frequently observed in plants under various biotic stress conditions. However, the mechanisms underlying the turning off of the photosynthetic activity and whether this process contributes to plants’ defense against pathogens remain to be determined. In this study, we analyze these mechanisms in  Plant defense against invading pathogens relies on a two-layered innate immune system. The first is the sensing of pathogen/microbe-associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs), which induces a basal level resistance known as PAMP-triggered immunity (PTI) –5. The sAll identified PRRs are membrane-localized receptor-like protein kinases (RLKs) or receptor-like proteins (RLPs) that detect conserved PAMPs such as bacterial flagellin and lipopolysaccharide or chitin from fungal cell walls ,12. PathPseudomonas syringae pv maculicola 1 (RPM1), Resistance to Pseudomonas syringae 2 (RPS2), and Resistance to Pseudomonas syringae 5 (RPS5) trigger downstream defense responses similar to that activated by PRRs during PTI, with the exception of different magnitude and duration pyrimidine (NA-PP1), a specific inhibitor of the sensitized MPK6YG. As revealed by BN-PAGE analysis, short-term MPK6 activation failed to induce photosynthetic inhibition , Pst-AvrRpt2, a Pst strain carrying the avirulence effector recognized by RPS2 (AvrRpt2) effector gene, or Pst-hrcC−, a Pst strain carrying a mutation in hrcC gene. Pst-AvrRpt2 triggers both PTI and CNL-type RPS2-dependent ETI in Arabidopsis. Due to the lack of functional type-III secretion system, Pst-hrcC− cannot deliver effectors into plant cells and only induces PTI.To determine the involvement of MPK3/MPK6 in photosynthetic inhibition during plant immunity, we first measured the kinetics of photosynthetic parameters in wild-type plants infiltrated with DD plants, Pst-AvrRpt2 induced drastic reductions in Fv/Fm   and ETI, respectively, besides PTI. To determine whether ETI is sufficient to induce PSII inhibition, we utilized the DEX-inducible promoter-driven AvrRpt2 (GVG-AvrRpt2) transgenic plants  glycerol). Thylakoid proteins for immunoblot were prepared by adding 1/4 volume of 4× SDS sample buffer  to solubilized thylakoid preparations and boiled for 2 min. NtMEK2DD expression was detected by using anti-FLAG . Activation of MPK3 and MPK6 was detected by using anti-pTEpY . Expression of Fld was detected by anti-Fld . Anti-D1 antibody  was used for the detection of PSII core D1 protein. The blots were incubated with horseradish peroxidase-conjugated goat-anti-mouse or goat-anti-rabbit secondary antibodies , and the bands were visualized using an enhanced chemiluminescence kit (Perkin Elmer) according to the manufacturer’s instructions.Protein extraction and immunoblot were carried out as previously described . Total pEF-1a was used as an internal control. The primer pairs used for qPCR are listed in Real-time quantitative PCR (qPCR) was performed as previously described . Total RDD seedlings treated with 2 μM DEX for 0 and 6 h, respectively. After DNase treatment, total RNA was purified using RNA clean and concentrator Kit. RNA sequencing libraries were constructed using TruSeq RNA library preparation Kit and sequenced using the HiSeq X Ten according to the manufacturer’s instructions. Dirty raw reads were filtered out using SONPnuke software. Clean reads were mapped to the Arabidopsis reference genome with BWA and to reference gene sequences with Bowtie. Gene expression levels were calculated using the RPKM method (reads per kb per million reads). The raw Illumina reads generated from RNA-seq experiments were deposited at NCBI Sequence Read Archive (SRP111959).Total RNA was extracted with TRizol reagent (Invitrogen) from 12-d-old p-values were indicated in figure legends. Each experiment was performed at least twice, with similar results.Statistical details of experiments are reported in figure legends. Statistical significance between groups was determined by one-way ANOVA comparison; S1 FigDD plants grown in liquid medium were treated with 5 μM DEX for indicated periods of time. Thylakoid membrane samples equivalent to 8 μg of chlorophyll content were subjected to first dimension BN-PAGE. The BN-PAGE strips were cut out. After denaturation, the samples were subjected to second dimension SDS-PAGE. Protein spots were visualized by brilliant blue G250 staining. Bands and protein spots were labeled as described previously. BN-PAGE, blue native polyacrylamide gel electrophoresis; CP43, photosystem II chlorophyll protein at 43 kDa; DD, GVG-NtMEK2DD; DEX, dexamethasone; LHCII, light-harvesting complex II; mc, mega-complex; MPK, mitogen-activated protein kinase; PSII, photosystem II; sc, super-complex; SDS-PAGE, sodium dodecyl sulfate-PAGE.Activation of MPK3/MPK6 causes disassembly of PSII-LHCII super-complexes and accumulation of the intermediate CP43-less PSII core complex. Twelve-d-old (TIF)Click here for additional data file.S2 Fig2O2 (A) and B) levels in whole seedlings. DD plants grown in liquid medium were treated with EtOH or 5 μM DEX and were kept under light for indicated periods of time. H2O2 and DD, GVG-NtMEK2DD; DEX, dexamethasone; EtOH, ethanol; MPK, mitogen-activated protein kinase; NBT, nitroblue tetrazolium; ROS, reactive oxygen species.Activation of MPK3/MPK6 leads to increases in H(TIF)Click here for additional data file.S3 FigDD and Col-0 plants grown in liquid medium were treated with 5 μM DEX and 200 nM flg22 for indicated times, respectively. Thylakoid membranes were isolated and solubilized with 1% dodecyl maltoside. Samples equivalent to 8 μg of chlorophyll were loaded to a BN-PAGE. For detection of D1 abundance in different complexes, samples equivalent to 2 μg of chlorophyll were loaded to a BN-PAGE. After transferring to a PVDF membrane, anti-D1 was used to detect D1 abundance. BN-PAGE, blue native polyacrylamide gel electrophoresis; Col-0, Columbia-0; DEX, dexamethasone; flg22, a 22 amino acids flagellin fragment; MPK, mitogen-activated protein kinase; PSII, photosystem II; PVDF, polyvinylidene fluoride.Twelve-d-old (TIF)Click here for additional data file.S4 FigA and B) Photosynthetic parameters are differentially affected by different Pst strains. Four-wk-old Col-0 plants were infiltrated with 10 mM MgCl2 (mock), Pst-EV, Pst-AvrRpt2, or Pst-hrcC− (OD600 = 0.2). The plants were covered with a transparent lid (100% relative humidity) and kept under light. Fv/Fm and Y(II) were measured at the indicated time. Values are means ± SD, n = 8. (C) Flg22-triggered PTI does not induce photosynthetic inhibition. Four-wk-old Col-0 plants were infiltrated with 10 mM MgCl2 (mock) or 50 nM flg22. Photosynthetic parameters were measured at 24 hpi. Values are means ± SD, n = 8. (D and E) AvrRpm1-triggered ETI also induces photosynthetic inhibition. Four-wk-old Col-0 plants were infiltrated with 10 mM MgCl2 (mock), P. fluorescens-EV (Pf0-1-EV), or P. fluorescens-AvrRpm1 (Pf0-1-AvrRpm1) (OD600 = 0.02). Fv/Fm and Y(II) were measured at indicated time points. Values are means ± SD, n = 8. The numerical values used to construct panels A–E can be found in AvrRpt2, avirulence effector recognized by RPS2; Col-0, Columbia-0; ETI, effector-triggered immunity; EV, empty vector; flg22, a 22 amino acids flagellin fragment; hpi, hours post inoculation; hrcC−, outer membrane type III secretion protein HrcC mutant; OD, optical density; Pf0-1-AvrRpm1, P. fluorescens-AvrRpm1; Pf0-1-EV, P. fluorescens-EV; Pst, Pseudomonas syringae pv tomato; PTI, PAMP-triggered immunity.((TIF)Click here for additional data file.S5 Fig2O2 production in chloroplasts. Twelve-d-old GVG-AvrRpt2/RPS2 and GVG-AvrRpt2/rps2 plants were treated with DEX (5 μM) or EtOH solvent control for 8 h. 2O2 accumulation was detected by NBT (A) and DAB (B) staining, respectively. Images of ROS accumulation at whole leaf level and subcellular level were shown. AvrRpt2, avirulence effector recognized by RPS2; DAB, 3,3′-diaminobenzidine; DEX, dexamethasone; ETI, effector-triggered immunity; EtOH, ethanol; GVG-AvrRpt2, DEX-inducible promoter-driven AvrRpt2; NBT, nitroblue tetrazolium; RPS2, Resistance to Pseudomonas syringae 2.AvrRpt2-mediated ETI results in light-dependent increases in (TIF)Click here for additional data file.S6 FigA) Four-wk-old plants were first sprayed with DMSO or 10 μM NA-PP1 for 2 h before infiltration with Pst-AvrRpm1 (OD600 = 0.0005), Pst-AvrB (OD600 = 0.0005), or Pst-AvrRps4 (OD600 = 0.0005). NA-PP1 or DMSO was sprayed again at 1.5 dpi. Pst-AvrRpt2 growth was quantified at 3 dpi. Values are means ± SD, n = 3, *P ≤ 0.001. (B) Plants pretreated with DMSO or 10 μM NA-PP1 were infiltrated with Pst-AvrRpm1 (OD600 = 0.02), Pst-AvrB (OD600 = 0.02), or Pst-AvrRps4 (OD600 = 0.02). Soil-grown Col-0, MPK6SR, and MPK3SR plants were first spray treated with 10 μM NA-PP1 for 2 h, and leaf discs were punched and then infiltrated with Pst-AvrRpt2 (OD = 0.02) by vacuum. Leaf discs were then transferred to GC vials containing 2 μM NA-PP1 or DMSO. Ion leakage was measured as increase in conductivity. Values are means ± SD, n = 3. (C) Four-wk-old soil-grown Col-0, MPK6SR, and MPK3SR plants were first spray treated with NA-PP1 (10 μM) or DMSO solvent control for 2 h and then infiltrated with Pst-AvrRpm1 (OD = 0.02), Pst-AvrB (OD = 0.02), and Pst-AvrRps4 (OD = 0.02). Values are means ± SD, n = 8. The numerical values used to construct panels A–C can be found in AvrB, avirulence protein B; AvrRpm1, avirulence effector recognized by RPM1; AvrRps4, avirulence effector recognized by RPS4; CNL, coiled coil-nucleotide binding site-leucine rich repeat; Col-0, Columbia-0; ETI, effector-triggered immunity; GC, gas chromatography; MPK, mitogen-activated protein kinase; NA-PP1, 4-amino-1-tert-butyl-3-(1’-naphthyl)pyrazolopyrimidine; NLR, nucleotide-binding leucine-rich repeat; OD, optical density; Pst, Pseudomonas syringae pv tomato; TNL, toll/interleukin-1 receptor-nucleotide binding site-leucine rich repeat.((TIF)Click here for additional data file.S7 FigA) Expression of pFld causes growth retardation. Four-wk-old soil-grown pFld transgenic plants in GVG-AvrRpt2 background were shown. (B) Immunoblot analysis of Fld expression in pFld transgenic plants using anti-Fld antibody. (C) AvrRpt2-induced PSII inactivation is delayed in pFld transgenic lines. Twelve-d-old GVG-AvrRpt2, GVG-AvrRpt2/pFld-4, and GVG-AvrRpt2/pFld-34 plants grown in liquid medium were treated with EtOH or 5 μM DEX. Fv/Fm was measured at 18 hpi. Values are means ± SD, n = 6, **P ≤ 0.001. The numerical values used to construct panel C can be found in GVG-AvrRpt2, DEX-inducible promoter-driven AvrRpt2; hpi, hours post inoculation; pFld, plastid-targeted cyanobacterial flavodoxin; PSII, photosystem II.((TIF)Click here for additional data file.S8 FigDD and DD pFld plants grown in liquid medium were treated with Pst-AvrRpt2 (OD = 0.02); disassembly of photosynthetic complexes at 24 hpi was visualized with BN-PAGE. Thylakoid membranes were isolated and solubilized with 1% dodecyl maltoside. Samples equivalent to 8 μg of chlorophyll were loaded to a blue native polyacrylamide gel (BN-PAGE). AvrRpt2, avirulence effector recognized by RPS2; BN-PAGE, blue native polyacrylamide gel electrophoresis; DD, GVG-NtMEK2DD; hpi, hours post inoculation; OD, optical density; pFLD; plastid-targeted cyanobacterial flavodoxin; PSII, photosystem II; Pst, Pseudomonas syringae pv tomato.Twelve-d-old (TIF)Click here for additional data file.S9 FigFld from cyanobacterium Anabaena sp. PCC 7119 was optimized using OptimumGene algorithm . Fld, flavodoxin.The coding sequence of (TIF)Click here for additional data file.S1 TableMPK, mitogen-activated protein kinase.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.