BACKGROUND: microRNAs (miRs) regulate the expression of protein-coding genes and play key roles in various biological processes, including development and immunity. However, dysregulation of miR expression is also involved in disease biology, including cancer.in vitro studies.METHODS: We utilized The Cancer Genome Atlas (TCGA) and other publicly available databases for miRs and mRNA expression in prostate cancer, selected miR-484 and investigated its role in prostate cancer biology and disease progression using in vitro assays with cancer cell lines showed that PSMG1 knockdown rescued the reduction in mobility brought on by miR-484 inhibition, pointing toward the existence of a miR-484–PSMG1 axis in prostate cancer.RESULTS: Our data mining efforts revealed that increased miR-484 in prostate tumors associates with early disease recurrence, while miR-484 expression in human prostate cancer cells enhances cancer cell mobility. Using RNAseq and bioinformatics, we identified candidate target genes of miR-484 and generated a list of potential tumor suppressors. One candidate in this list was PSMG1. We applied luciferase assays and immunoblotting to confirm that miR-484 directly targets PSMG1. Additional CONCLUSIONS: We hypothesize that miR-484 is an oncogene in the prostate that increases cancer cell mobility, with PSMG1 being a mir-484 target in this process. Caenorhabditis elegans [Since their initial discovery in  elegans , microRN elegans  and anim elegans . miRs re elegans , which lAn area of interest among researchers is the implication of miRs in diseases. For instance, miR-33 deficiency in mice has been noted to promote hyperlipidemia and obesity, but its deficiency in macrophages decreased lipid accumulation, reducing atherosclerotic plaques . In anotOur laboratory has studied prostate carcinogenesis and identified miRs that are involved in disease progression. For instance, miR-1 is a candidate tumor suppressor , as its The following six human prostate cell lines were obtained from the ATCC : RWPE-1, RWPE-2, 22Rv1, LNCaP, DU145, and PC-3. These cell lines have been regularly authenticated using a short tandem repeat analysis with GenePrint10 and tested for Mycoplasma contamination.All reagents and equipment were from Thermo Fisher Scientific . Total RNA was isolated using TRIzol (Cat# 15596026) according to instructions.Ten nanograms of total RNA were reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit (Cat# 4366596). miR-qPCR assessing expression levels of miR-484 was performed in triplicate using TaqMan probes for miR-484  and U6 snRNA  with TaqMan Universal Master Mix II, no UNG (Cat# 4440041). The U6 snRNA was used as the internal standard reference.CT 13-20.A thousand nanograms of total RNA were reverse transcribed using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Cat# 4374967). qPCR was performed in triplicate using TaqMan probes for PSMG1 , TSPAN15 , SNRNP200 , USP18  and 18s rRNA  with TaqMan Gene Expression Master Mix (Cat# 4369016). The 18s rRNA was used as the internal standard reference. Owing to the high abundance of 18s rRNA, total RNA was diluted 1:100 for the 18 s rRNA assay yielding average CT method, and fold changes were calculated from 2CTΔΔ values for miR-484 or each gene [Assays were run on MicroAmp Optical 384-Well Reaction Plate with Barcode (Cat# 4343814) and analyzed using 7900HT Fast Real-Time PCR System with 384-well block module (Cat# 4329001). Normalized expression was calculated using the comparative ach gene .5 cells/well in six-well plates. Twenty-four hours after seeding, cells were transfected with 30 nM of mirVana miRNA inhibitor, negative control #1 (Cat# 4464076), or mirVana miR-484 inhibitor  using Lipofectamine RNAiMAX (Cat# 13778150) from Thermo. Forty-eight hours after transfection, four columns of wells in a 96-well plate were seeded with each of the following: 100 μl of media/well with no cells, 1 × 103 negative control-transfected cells/well or 1 × 103 miR-484 inhibitor-transfected cells/well. Each of the four columns of wells represented eight replicates for a time point—Day 0, 1, 3 or 5. Normalized expression of miR-484 was calculated using the comparative CT method, and fold changes were calculated from 2CTΔΔ values for miR-484. A 70–80% reduction of miR-484 induced by the inhibitor relative to the negative control was verified by miR-qPCR.Human prostate cells were seeded at 2 × 10t test with Welch’s correction in GraphPad Prism 7. The experiment was repeated for n = 3.Cell viability was measured on Days 0, 1, 3 and 5 using alamarBlue Cell Viability Reagent (Cat# DAL1025) from Thermo with a FLUOstar Omega microplate reader from BMG Labtech , using fluorescence (excitation at 544 and emission at 590 nm). Background fluorescence values of media-only wells were subtracted from the values of wells with negative control or miR-484 inhibitor-transfected cells. The subtracted fluorescence values of the eight replicates from negative control or miR-484 inhibitor-transfected cells on Days 0, 1, 3 and 5 were compared using unpaired 6 cells/well in T25 flasks with n = 3. Twenty-four hours after seeding, cells were transfected with 30 nM of mirVana miRNA inhibitor, negative control #1 (Cat# 4464076) or mirVana miR-484 inhibitor  using Lipofectamine RNAiMAX (Cat# 13778150) from Thermo. Forty-eight hours after transfection, 1.2 × 106 cells were harvested, and the cell pellets frozen at −80°C. Normalized expression of miR-484 was calculated using the comparative CT method, and fold changes were calculated from 2ΔΔCT values for miR-484. A 70–80% reduction of miR-484 induced by the inhibitor relative to the negative control was verified by miR-qPCR.Cells were seeded at 1.6 × 10t test with Welch’s correction in GraphPad Prism 7.The caspase-3 activity, a readout of apoptosis, was determined in cell pellets with EnzChek Caspase-3 Assay Kit #2 (Cat# E13184) from Thermo and measured using a FLUOstar Omega microplate reader from BMG Labtech, using fluorescence (excitation at 485 and emission at 520 nm). The caspase-3 values of negative control or miR-484 inhibitor-transfected cells were compared using unpaired 5 cells/well in six-well plates. Two different transfection conditions were performed as follows:mirVana miRNA inhibitor, negative control #1 (Cat# 4464076), or mirVana miR-484 inhibitor  using Lipofectamine RNAiMAX (Cat# 13778150) from Thermo to assess the effect of miR-484 inhibition. Forty-eight hours after transfection, normalized expression of miR-484 was calculated using the comparative CT method, and fold changes were calculated from 2CTΔΔ values for miR-484. A 70–80% reduction of miR-484 induced by the inhibitor relative to the negative control was verified by miR-qPCR.Twenty-four hours after seeding, cells were transfected with 30 nM of mirVana miRNA inhibitor, negative control #1 (Cat# 4464076) and Silencer Select negative control #1 siRNA (Cat# 4390843); (2) 30 nM each of mirVana miR-484 inhibitor  and Silencer Select negative control #1 siRNA (Cat# 4390843); or (3) 30 nM each of mirVana miR-484 inhibitor  and PSMG1 Silencer Select siRNAs  using Lipofectamine RNAiMAX (Cat# 13778150) from Thermo to assess the effect of miR-484 and PSMG1 dual inhibition. Forty-eight hours after transfection, normalized expression of PSMG1 was calculated using the comparative CT method, and fold changes were calculated from 2CTΔΔ values for PSMG1. Downregulation of miR-484 and upregulation of PSMG1 in response to mirVana miRNA inhibitor transfection were verified by qPCR. Expression differences were compared with Silencer Select negative control siRNA.Twenty-four hours after seeding, cells were transfected with the following: (1) 30 nM each of Cells were seeded at 2 × 104 cells for each treatment condition were seeded in replicate wells in the upper chamber of CIM plate 16 (Cat# 05665817001), whose bottom chamber contained media with 10% FBS as an attractant. The assembled chambers of CIM plate 16 were loaded onto xCELLigence RTCA Systems (Cat# 00380601050) from ACEA Biosciences , and cell movements from upper to bottom chambers were quantified in 15-min intervals for 48 h. The experiment was repeated for n = 3.For migration assay, 3 × 10n = 3. We used Matrigel at a 5% concentration.For invasion, the steps of migration assay were replicated with an additional step 48 h after transfection: the wells in the upper chamber of CIM plate 16 were coated with Matrigel Matrix (Cat# 356234) from BD Biosciences , and the upper chamber was placed in a 37°C incubator for 4 h for the Matrigel to solidify. The experiment was repeated for mirVana miRNA mimic, negative control #1 (Cat# 4464058), or miR-484 mimic  from Thermo . Forty-eight hours after transfection, total protein was extracted from cells using RIPA buffer (Cat# 89901) with Halt Protease Inhibitor Cocktail (Cat# 87786), and 50 μg of protein was loaded on a 16% Tris-Glycine gel (XP00165BOX) from Thermo followed by immunoblotting. Primary antibodies used were PSMG1 (1:1000 of Cat# 13378) from Cell Signaling  and β-Actin (1:5000 of sc-47778) from Santa Cruz . The secondary antibody for the PSMG1 Western was an ECL anti-Rabbit IgG, HRP-linked F(ab')2, fragment (from donkey) (#NA9340V) from GE Healthcare , used at a 1:5000 dilution. Visualization of β-actin using the labeled primary antibody from Santa Cruz does not require a secondary antibody. Chemiluminescent signal was captured on the Bio-Rad ChemiDoc Imaging System. The experiment was repeated for n = 3.DU145 cells were transfected with 30 nM of mirVana miRNA mimic, negative control #1 (Cat# 4464058), or miR-484 mimic  from Thermo in quadruplicates and submitted to RNAseq and analyzed to identify downregulated genes, the likely targets of the miR.DU145 and PC-3 cells were transfected with 100 nM of Ensembl database (v70) using the Tophat software. RNA mapping statistics were calculated using the Picard software, and the average uniquely aligned reads were approximately 90% for each sample. We subsequently used the RNA-Seq workflow module in Partek Genomics Suite 6.6 from Partek Inc.  and the R/Bioconductor package DESeq2 to identify differentially expressed genes at FDR < 5% and a fold change (≥1.5) cut-off. Partek performed an analysis based on RPKM units (reads per kilobase per million mapped reads), and the gene counts were fitted to a negative binomial generalized linear model with DESeq2.RNAseq was performed at the Sequencing Facility, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research . Briefly, 500 ng of total RNA was used for library preparation with the TruSeq V3 chemistry kit from Illumina . Sequencing was performed on an Illumina HiSeq 2000 system. For each sample, we generated approximately 50 million paired-end read at 101 bp length. Reads were trimmed for both adapters and low-quality bases using the Trimmomatic software and then aligned with the reference human hg19 genome and gene annotation from the http://www.targetscan.org) was utilized to generate a list of predicted gene targets of miR-484.The TargetScan database  Venny software (http://bioinfogp.cnb.csic.es/tools/venny/index.html) was applied, which is an interactive tool for comparing gene lists with Venn diagrams.To generate an overlap between the downregulated transcripts in miR-484 mimic-transfected DU145 or PC-3 cells . The 49 and 42 candidate genes from the DU145 and PC-3 gene lists, respectively, that overlapped with predicted TargetScan targets were then further compared with the list of genes from these eight cohorts; the ones showing overlap with genes that had greater expression in normal tissue versus tumor in ≥ 2 cohorts were selected and searched for using PubMed. By doing so, we aimed to further narrow down the list of potential tumor suppressors targeted by miR-484.In order to narrow down the list of genes from the overlap, expression of genes in prostate tumor versus non-cancerous tissues from eight cohorts  was utilized to locate the putative binding site of miR-484 in PSMG1 3′ UTR. The information was used to generate pLenti-UTR-Luc PSMG1 WT and pLenti-UTR-Luc PSMG1 MUT for the luciferase assays.The TargetScan database  from abm . The pieces were joined using the Ligation-Free Cloning kit, inserting each 3′ UTR into the reporter construct. The sequences of constructs were verified prior to experiments.The reporter construct, pLenti-UTR-Luc, consists of a CMV promoter followed by luciferase from TCTTAAACATTGTTTTGTAGTGTATATTACTTGTCCATTCCTTTAAGGGGAGCAGCCTGCACTCTTTTGTAGATTACTTTTGGGGGATATATTTTGAGAATGATGAAACGGAATAAAATTGTAAAAAATTAATTGTAGTTTTAA.TTTAAACACTCTTTTGTAGATTACTTTTGGGGGATATATTTTGAGAATGATGAAACGGAATAAAATTGTAAAAAATTAATTGTAGTTTTAA.TCTTAAACATTGTTTTGTAGTGTATATTACTTGTCCATTCCTTTAAGGGGAGCmirVana miRNA mimic, negative control #1 (Cat# 4464058), or miR-484 mimic  from Thermo, 100 ng of luciferase reporter pLenti-UTR-Luc PSMG1 WT or pLenti-UTR-Luc PSMG1 MUT, and 4 ng of pRL-CMV Renilla luciferase reporter (Cat# E2261) from Promega , using TransIT-X2 (Cat# 6004) from Mirus . Cells were cultured for another 48 h and washed once in PBS. Using a Luc-Pair Duo-Luciferase HS Assay Kit (Cat# LF004) from GeneCopoeia , cells were then lysed, loaded onto white 96-wells in quadruplicates, and their luciferase/Renilla ratios were measured using a FLUOstar Omega microplate reader from BMG Labtech. The experiment was repeated for n = 4.Seventy-thousand cells were seeded in 24-well plates and co-transfected with 100 nM of t test with Welch’s correction from GraphPad Prism 7.0 was used to assess the endpoints, including differences in miRNA and mRNA expression, apoptosis and luciferase assays. All statistical tests were two-sided, and P < 0.05 was considered significant. Kaplan–Meier analysis in GraphPad Prism 7.0 was utilized for comparing differences in disease-free survival between groups of patients divided by miR-484 or PSMG1 expression.Unpaired http://cbio.mskcc.org/cancergenomics/prostate/data/) to obtain expression of miR-484 between tumor and normal tissues of patients. Data from Taylor et al. Cancer Cell 2010 were downloaded from the NCBI and utilized to obtain miR-484 expression, PSMG1 expression and disease-free survival of patients according to the expression of miR-484 and PSMG1.TCGA data on prostate cancer were downloaded from the cBio Cancer Genomics Portal (We hypothesized that the miRs of interest in prostate carcinogenesis are the ones significantly related to survival of patients. Therefore, we assessed that miRs have the most robust relationships with survival in TCGA and Taylor datasets and identified miR-484 as being significantly associated with decreased disease-free survival A. This wWe assessed miR-484 expression by miR-qPCR in six human prostate cell lines A and notIn order to identify protein-coding gene targets of miR-484 in prostate cancer cells beyond the prediction provided by TargetScan, we transfected two human prostate cancer cell lines, DU145 and PC-3, with a miR-484 mimic and searched for the genes commonly downregulated by this approach using RNAseq analysis of miR-484 mimic and negative control miR-transfected cells. We chose DU145 and PC-3 cells because they are both derived from metastases and express the highest and the lowest level of miR-484 among the three metastatic cell lines A. By oveAfter analyzing the RNAseq data from the miR mimic-transfected cells, we then overlapped the list of downregulated genes with the list of predicted gene targets of miR-484 in TargetScan . We subsThe search for gene targets of miR-484 rendered several candidates, one of which was PSMG1; it showed the most significant change and had the greatest fold decrease . This geIn order to confirm that PSMG1 is a direct target of miR-484, we transfected DU145 with miR-484 mimic and luciferase plasmids, which contained either the unaltered or mutated binding site of the miR in PSMG1 3′ UTR, and assayed luciferase activity A. We detWe wanted to know whether miR-484 inhibition would reduce cancer cell proliferation. The reduction would confirm the hypothesized tumor suppressive role of PSMG1  in reducWe lastly examined whether miR-484 may influence cancer cell migration and invasion, which are essential traits of metastasis. We found that miR-484 inhibition significantly reduced migration and invasion C,D. We fmiRs can act as oncogenes by directly targeting tumor suppressors, consequently downregulating them. An example of such includes miR-21, which acts as an oncogene in colon cancer by inhibiting Pdcd4 and consequently increasing invasion and metastasis . AnothermiR-484 has been reported to be active in a variety of conditions. For instance, it targets PCDH19 to decrease proliferation but increases neurogenesis in mice . miR-484In a meta-analysis of six miR datasets in prostate cancer, miR-484 was upregulated, which indicated it might be a negative prognostic biomarker . In contIn TCGA, we found that miR-484 is highly expressed in tumors compared with non-cancerous tissues, and it was inversely associated with survival in a publicly available database , which sWe found several reports stating that PSMG1 increases susceptibility for inflammatory bowel diseases , which ain vitro experiments using cancer cell lines, which have limitations. Therefore, future murine in vivo data showing clear differences in metastasis brought on by miR-484 modulation would strengthen the conclusions. In addition, elucidation of functions of PSMG1, other than proliferation and mobility, relevant to carcinogenesis would complement the current findings and establish the importance of miR-484–PSMG1 axis in prostate cancer.While the present work shows that miR-484 is a candidate negative prognostic biomarker in prostate cancer likely by targeting PSMG1 and modulating cell mobility, its conclusions are based on analyses from patient samples and Click here for additional data file.


GmNFYA13, an NF-YA gene in soybean, was strongly induced by salt, drought, ABA, and H2O2, and suppressed by tungstate, an ABA synthesis inhibitor. The GmNFYA13 transcripts were detected in different tissues in seedling and flowering stages, and the expression levels in roots were highest. GmNFYA13 is a nuclear localization protein with self-activating activity. Transgenic Arabidopsis plants overexpressing GmNFYA13 with higher transcript levels of stress-related genes showed ABA hypersensitivity and enhanced tolerance to salt and drought stresses compared with WT plants. Moreover, overexpression of GmNFYA13 resulted in higher salt and drought tolerance in OE soybean plants, while suppressing it produced the opposite results. In addition, GmNFYA13 could bind to the promoters of GmSALT3, GmMYB84, GmNCED3, and GmRbohB to regulate their expression abundance in vivo. The data in this study suggested that GmNFYA13 enhanced salt and drought tolerance in soybean plants.NF-YA transcription factors function in modulating tolerance to abiotic stresses that are serious threats to crop yields. In this study,  As types of osmotic stress, salt and drought stresses are major challenges to growth and development of plants, resulting in severe yield losses . Many chNFYA5  cultivar Williams 82 were grown in plastic pots  containing nutrient soil and grown for 20 days in a tissue culture room at 28/18°C day/night with a 14-h photoperiod and 70% relative humidity as described previously  could affect salt-induced GmNFYA13 transcripts, two detached leaves of soybean plants were placed into ddH2O to eliminate the effect of wound stress for 1 h, and then transferred into ddH2O and 2 mM tungstate for 3 h, followed by being subjected to 200 mM NaCl solution for 1 h. To determine whether tungstate could suppress the drought-induced expression level of GmNFYA13, the “drought + tungstate” treatment was performed as described previously  and primer set 5′-GGTAGTCAATAGTCATCACTT-3′ and 5′-CAAGTTGGATGAGATAAAGCC-3′. The PCR products were cloned into the vector pEASY-Blunt  and sequenced; the correct clone was stored at −80°C for isolation of the CDS of GmNFYA13.Williams 82 soybean plants were used for RNA isolation, followed by reverse transcription to cDNA. The full-length coding sequence (CDS) of NcoI site to generate the pCAMBIA1302:GmNFYA13 fusion construct, which is driven by the promoter of cauliflower mosaic virus 35S (CaMV35S). The primer set used in this assay was 5′-GGGACTCTTGACCATGATGCCAGGGAAACCT-3′ and 5′-TCAGATCTACCATGGCTTTGAAATCCCCATT-3′. The pCAMBIA1302:GmNFYA13 fusion constructs were transformed into Agrobacterium tumefaciens strain GV3101, followed by infection of Arabidopsis flowers. Hygromycin  was used to screen the positive transgenic lines to obtain the T3 homozygotes. The 35S:GmNFYA13-2, 35S:GmNFYA13-4, and 35S:GmNFYA13-5 transgenic Arabidopsis lines were used to explore the function of GmNFYA13.The CDS without the termination codon was fused in pCAMBIA1302 vector and ligated with 35S:GmNFYA13 and WT Arabidopsis plants on 1/2 MS medium were used to determine the localization of GmNFYA13 in cells. Roots of 35S:GmNFYA13 plants could express GFP-GmNFYA13 fusion protein. Roots of 35S:GmNFYA13 and WT Arabidopsis plants were detached and observed using a confocal laser scanning microscope  as described previously  with the primer set 5′-GGACACGTGTCTCGATCTTCG-3′ and 5′-CTTAGCACAACTTTTCCCTAT-3′. The products were cloned into a modified pCAMBIA1305 vector to generate the pCAMBIA1305:PGmNFYA13 fusion construct with the forward primer 5′-CCATGATTACGAATTCGGACACGTGTCTCGATCTTCG-3′ and reverse primer 5′-CTCAGATCTACCATGGCTTAGCACAA CTTTTCCCTAT-3′ as previously described  for 2 weeks. For salt treatment, the 3-week-old seedlings were irrigated with 350 mM NaCl solution; for drought treatment, water supply was shut off until significant difference was detected between 35S:GmNFYA13 transgenic Arabidopsis plants, the leaves of 3-week-old seedlings were collected at 1 h after salt treatment. To assess the transcription levels of drought-related genes in 35S:GmNFYA13 lines, the leaves of 3-week-old seedlings were sampled at 2 h after drought treatment. The collected leaves were immediately frozen with liquid nitrogen for RNA isolation. The leaves of 3-week-old seedlings under normal conditions were set as the control.To assess the transcription levels of salt-responsive genes in the GmNFYA13, the CDS of GmNFYA13 without the termination codon was introduced into the vector pCAMBIA3301 to generate the pCAMBIA3301:GmNFYA13 construct driven by the CaMV35S promoter and ligated with NcoI and BstEII sites with the primer set 5′-GGACTCTTGACCATGATGCCAGGGAAACCTGAC-3′ and 5′-ATTCGAGCTGGTCACCTTTGAAATCCCCATTAGG-3′. To generate transgenic soybean plants suppressing GmNFYA13, a 705-bp RNA interference fragment (GmNFYA13 construct. The pCAMBIA3301:GmNFYA13 (OE), pCAMBIA3301:RNAi-GmNFYA13 (RNAi), and pCAMBIA3301 empty vector (EV) were transformed into A. rhizogenes K599 and stored at −80°C.To generate transgenic soybean plants overexpressing fragment  was artiA. rhizogenes K599 harboring OE, RNAi, and EV construct with a syringe tip, and then covered by plastic cups for 24 h without light. The soybean plants were uncovered, and the infected hypocotyls were surrounded by peat and vermiculite mixture . They were irrigated with enough water to create a warm and humid environment for 2 weeks, and then hairy roots were induced. The principal roots were cut off, and soybean plants with positive hairy roots screened by QuickStix kit  and qRT-PCR were used to perform subsequent experiments; they were, respectively, named OE, RNAi, and EV transgenic soybean plants.Soybean seeds were sown into nutrient soil in plastic pots  and the hypocotyls of 5-day-old seedlings were infected by The transgenic soybean plants with positive hairy roots were planted in plastic pots  containing nutrient soil for 7 days to establish normal growth. It is difficult to complete the entire growth period using plants with only a few positive hairy roots under salt and drought stresses. However, the transgenic soybean plants are sufficient for drought tolerance at the seedling stage.For salt treatment, the soybean plants were irrigated with 500 mM NaCl solution; for drought treatment, the water supply to soybean plants was shut off until significant difference was observed among the three transgenic soybean lines.To evaluate the transcription level of salt/drought-related genes in transgenic soybean plants, for salt and drought treatments, the positive hairy roots were sampled at 1 h after salt stress and 2 h after drought stress. The control was the non-treated positive hairy roots.CT–ΔΔ method based on CT values  with a 16-h photoperiod and 50–60% relative humidity as described previously . TobaccoGmSALT3 was 5′-GTCGACGGTATCGATAAGCTTCGATGATACTACATAAGT-3′ and 5′-CAGGAATTCGATATCAAGCTTGGCCAAAGACTC AGTGCT-3′, with 5′-GTCGACGGTATCGATAAGCTTCCTGG TCCACTCTAGTAG-3′ and 5′-CAGGAATTCGATATCAAGCT TGGTGAATGGTCGGTTGAA-3′ for GmNCED3. Primers for GmMYB84 were 5′-GTCGACGGTATCGATAAGCTTCTCAG CTTCAATTACAAT-3′ and 5′-CAGGAATTCGATATCAAGCT TGGCTATTCTCACTCACTA-3′; primers for GmRbohB were 5′-GTCGACGGTATCGATAAGCTTACGACACAGTCTGAGAAT-3′ and 5′-CAGGAATTCGATATCAAGCTTCTCCTCGTCGTCC TTGAA-3′. The fusion constructs pGreen II 0800:PGmSALT3/GmNCED3/GmMYB84/GmRbohB were transformed into A. tumefaciens strain GV3101. GV3101 harboring the pCAMBIA1302 empty vector and pCAMBIA1302:GmNFYA13 fusion construct were co-transformed into tobacco leaves with GV3101 harboring pGreen II 0800:PGmSALT3/GmNCED3/GmMYB84/GmRbohB. The GUS fluorescence was observed using LB985 NightSHADE  following the manufacturer’s instructions.The promotors of target genes were inserted into pGreen II 0800 as previously described . The pri2, 10 mM EDTA, and 5 mM DTT, and then pH was adjusted to 8.0. The LUC expression levels of the protein solution were evaluated with GloMax Multi Jr  according to the manufacturer’s instructions.To quantify the LUC activity in the tobacco leaves of equal weight, the protein was extracted. The extraction buffer consisted of 50 mM Tris, 0.5 M sucrose, 1 mM MgClt-test. Three biological replications were performed for each experiment in this study.The data in this research were subjected to Duncan’s tests using SPSS Statistics 22 . The data of germination rate, water loss, and SWP were assessed with Student’s GmNFYA5 was the highest among all 21 GmNFYAs under drought conditions  is 912 bp in length, encoding a 34.06-KD polypeptide with an isoelectric point (pI) of 9.50. The conserved core regions of GmNFYA13, which consists of an NF-YB/C binding subdomain and CCAAT binding sequences, are similar to those of Arabidopsis  were used to carry out further assays based on RT-PCR analysis  (GmNCED3)   , one keyGmNCED3) , three dGmMYB84) , and onee stress , were seGmNCED3 among OE, EV, and RNAi soybean plants . Arabidopsis gene sequences in this study were downloaded from The Arabidopsis Information Resource (TAIR) (http://www.arabidopsis.org/). The gene accessions are listed in Soybean gene sequences in this study were downloaded from the plant genomics resource (Z-SX coordinated the project, conceived and designedexperiments, and edited the manuscript. X-JM performed experiments and wrote the first draft. J-DF and Y-MT conducted the bioinformatic work and performed experiments. T-FY, Z-GY, JC, Y-BZ, MC, and Y-ZM provided analytical tools and managed reagents, coordinated the project, and contributed valuable discussions. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The immunogenicity and induced protective immunity of MAP against Trichinella spiralis  infection were evaluated in mice. We demonstrated that mice immunized with MAP containing CD4+ T cell and B cell epitopes (MAP-TB) induced significantly higher protection against the challenge of T. spiralis larvae (35.5% muscle larva reduction) compared to the MAP containing B cell epitope alone (MAP-B) with a 12.4% muscle larva reduction. The better protection induced by immunization of MAP-TB was correlated with boosted antibody titers (both IgG1 and IgG2a) and mixed Th1/Th2 cytokine production secreted by the splenocytes of immunized mice. Further flow cytometry analysis of lymphocytes in spleens and draining lymph nodes demonstrated that mice immunized with MAP-TB specifically enhanced the generation of T follicular helper (Tfh) cells and germinal center (GC) B cells, while inhibiting follicular regulatory CD4+ T (Tfr) cells and regulatory T (Treg) cells. Immunofluorescence staining of spleen sections also confirmed that MAP-TB vaccination enhanced the formation of GCs. Our results suggest that CD4+ T cell epitope of Ts-Pmy is crucial in vaccine component for inducing better protection against T. spiralis infection.Multiepitope peptide vaccine has some advantages over traditional recombinant protein vaccine due to its easy and fast production and possible inclusion of multiple protective epitopes of pathogens. However, it is usually poorly immunogenic and needs to conjugate to a large carrier protein. Peptides conjugated to a central lysine core to form multiple antigen peptides (MAPs) will increase the immunogenicity of peptide vaccine. In this study, we constructed a MAP consisting of CD4 Trichinella spiralis [T. spiralis infection in pigs was 0.61% (5/823) in Henan Province of China, in which 0.91% (5/550) of pigs were infected in Nanyang city alone [Trichinella infection in China [T. spiralis from wild animal reservoirs to human beings [Trichinellosis is a worldwide food-borne zoonosis spread between animals and people and mainly caused by the infection of spiralis . People spiralis . In Chinspiralis . A pork ty alone . It has as 0.61% /823 in HT. spiralis infection with the purpose of reducing worm fecundity or decreasing muscle larval and adult worm burdens [T. spiralis has a complex life cycle, diverse stage-specific antigens, and immune-evasion strategies [In the past 30 years, many efforts have been dedicated to develop vaccine against  burdens . The vac burdens , recombi burdens , 9, and  burdens , inducinrategies , 12. Subrategies .T. spiralis (Ts-Pmy), has exhibited partially protective immunity against T. spiralis infection in mice [Ts-Pmy, YX1, has been characterized by screening a phage display peptide library with a protective monoclonal antibody (mAb) named as 7E2 [T. spiralis [+ T cell epitopes of Ts-Pmy identified previously [Ts-Pmy (YX1) to construct a multiple antigen peptide (MAP-TB) in this study. We have observed that mice immunized with this fused MAP (MAP-TB) based on T/B epitopes of Ts-Pmy induced potent protection against T. spiralis infection which is associated with enhanced humoral immune responses.In our previous study, a promising vaccine candidate, paramyosin (Pmy) of  in mice . A proted as 7E2 . In ordespiralis , two poteviously  were fusThis study was performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Experimental Animals. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Capital Medical University .d) mice were obtained from the Laboratory Animal Services Center of Capital Medical University  and raised under specific pathogen-free standard conditions. Each experimental group consisted of ten mice. T. spiralis (ISS 533) strain used in this study was maintained in female ICR mice, and the muscle larvae were recovered from the muscle of infected mice using a modified pepsin-hydrochloric acid digestion method as described by Gamble et al. [Six- to eight-week-old female BALB/c  fused with a B cell epitope (MAP-TB) or only B cell epitope (MAP-B). The T-B peptides are linked at their C terminus to the lysine core of the MAP. Two different CD4+ T cell epitopes, T2 and T5, representing the CD4+ T cell epitopes P2 and P5 identified in our previous studies [T. spiralis infection by passive transfer [MAP-TB and MAP-B constructed in this study are four-branched MAPs containing either two CD4 studies , 19, wertransfer . The MAPμg of MAP-TB or MAP-B (50 μl) was emulsified with the same volume of adjuvant for a single dose. Another 10 mice were subcutaneously injected with 50 μl PBS emulsified with the same volume of the corresponding adjuvant as the control. One week after the final boost, five mice from each group were sacrificed for collecting sera, spleens, and inguinal lymph nodes (ILNs) to evaluate the induced immune responses. The rest 5 mice were challenged with 400 infective muscle larvae (ML) of T. spiralis for evaluating the protection as described below. A representation of experimental design is shown in To evaluate the immune response induced by the MAP-TB and MAP-B, ten female BALB/c mice were subcutaneously immunized on the back. The MAPs were emulsified with Freund's adjuvant  using complete adjuvant for the initial dose and incomplete adjuvant for the following two booster inoculations at an interval of two weeks. 30 -B cell epitope-specific antibodies induced by MAP immunization were measured by ELISA. Briefly, the plate was coated with 10 μg/well of B cell epitope peptide YX1 in coating buffer overnight at 4°C. After blocking, serial dilutions of sera  were added at 37°C for 30 min followed with the HRP-conjugated goat anti-mouse IgG (1 : 1000 dilution). The titers of each group were expressed as the geometric mean of the dilution. For IgG subclass detection, the sera were diluted at 1 : 2000 and 1 : 5000, then incubated with Biotin-conjugated Rat Anti-Mouse IgG1 or IgG2a  followed by Streptavidin-HRP . The color was developed with tetramethylbenzidine substrate  and read at 450 nm.Anti7 cells/ml in complete RPMI-1640 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). For in vitro stimulation, a total of 1 × 106 splenocytes were incubated with mixed peptides  in 200 μl of complete RPMI-1640 for 48 h at 37°C in a humidified atmosphere containing 5% CO2. Splenocytes stimulated simultaneously with ConA  were served as positive controls. The cytokines IFN-γ, IL-2, IL-4, IL-5, and IL-6 were detected using the corresponding ELISA kit , according to the manufacturer's instructions.One week after the final immunization, mice were sacrificed and splenocytes were separated aseptically using mouse lymphocyte separation medium . After being centrifuged, the spleen cells were resuspended and adjusted to 1 × 10+PD-1+CD3+CD4+ Tfh cells (in spleens), CXCR5+PD-1highCD3+CD4+ Tfh cells (in ILNs), and CD25+FoxP3+CD3+CD4+ Treg cells (in spleens) presented within the total CD4+ T cells; the frequencies of GL7+Fas+B220+ germinal center (GC) B cells presented within the total B220+ B cells; and the frequencies of CXCR5+FoxP3+CD3+CD4+ Tfr cells presented within the CD4+ T cells (in spleens and ILNs) were detected. Dead cells were excluded by viability dye staining, and duplicates were excluded by FSC/A and FSC/W gating analysis. Cells were analyzed by a BD LSRFortessa™ Flow Cytometry . Data were acquired and analyzed by BD FACSDIVA™ version 8.0.2 .Spleens and ILNs of mice were harvested on day 7 after the final immunization. The lymphocytes were collected and analyzed by flow cytometry. The frequencies of CXCR5For surface staining, splenocytes were blocked with anti-CD16/CD32 mAb  and stained with the following antibodies: anti-CD3e-Percp-Cyanine5.5 , anti-CD4-FITC , anti-PD-1-PE , and anti-CXCR5-APC  for Tfh cell analysis; with anti-GL7-FITC , anti-Fas-PE , and anti-B220-PerCP-Cy5.5  mAb for GC B cell analysis. For viability dye staining, Fixable Viability Dye eFluor™ 450  was added with surface staining antibodies.For intracellularly staining (Treg/Tfr), the splenocytes were blocked with anti-CD16/CD32 mAb , stained with anti-CD3e-Percp-Cyanine5.5, anti-CD4-FITC and anti-CXCR5-APC, or anti-CD25-APC , then fixed with FoxP3 Fixation/Permeabilization working solution  and permeabilized with Permeabilization Buffer . After that, the cells were intracellularly stained with anti-FoxP3-PE .μm) on a Cryostat . After being fixed in cold acetone and blocked with 1% BSA in PBS at room temperature for 1 h, the sections were incubated with Biotinylated Peanut Agglutinin  at 4°C overnight. DyLight™ 488 Streptavidin  was used as the secondary antibody at room temperature for 1 h. At last, Alexa Fluor® 647-conjugated anti-mouse CD45R  was incubated at room temperature for 1 h. The sections were scanned under a Pannoramic SCAN instrument . For quantification the area of GCs, spleen sections of three mice from each group were analyzed by ImageJ software.The spleens from BALB/c mice were separated one week after the final immunization and embedded in Optimal Cutting Temperature (OCT) Compound . The tissues were frozen at -80°C before sectioning  muscle from the mice immunized with MAP-TB or MAP-B versus those from the PBS control group. Specifically, it was calculated as follows: worm burden reduction rate (%) = (1 − mean number of LPG in vaccinated mice/mean number of LPG in control mice) × 100%. Three independent experiments were carried out.One week after the third immunization, the remaining five mice of each group were challenged orally with 400 P value < 0.05 were considered statistically significant.Statistical analyses were performed with One-way ANOVA using SPSS for Windows, version 17.0. All data are expressed as the mean value + standard deviations (SD) or the mean value ± SD. P < 0.01). The IgG antibody subclass was determined at a 1 : 2000 dilution , Th2 cytokines (IL-4 and IL-5), and IL-6 in the culture supernatants of splenocytes upon stimulation were significantly elevated in mice immunized with MAP-TB as compared with mice immunized with MAP-B or PBS (The cytokines IFN-B or PBS . However+PD-1+CD3+CD4+ Tfh cells (in spleens), CXCR5+PD-1highCD3+CD4+ Tfh cells (in ILNs), GL7+Fas+B220+ GC B cells (in spleens), CXCR5+FoxP3+CD3+CD4+ Tfr cells [+FoxP3+CD3+CD4+ Treg cells (in spleens). Representative dot plots of flow cytometric analysis are shown in +PD-1highCD3+CD4+ T cells residing in lymph node GC represent a specific Tfh subset that correlate with B cell maturation and IgG production [+ T cells of spleens and ILNs and GC B cells within total B220+ B cells of spleens increased in mice immunized with MAP-TB compared with the MAP-B or PBS group , which was also significantly higher than MAP-B immunization . However, there was no significant difference in the ML burden between the MAP-B and PBS groups .Challenge experiments demonstrated that mice immunized with MAP-TB induced 35.5% ML reduction compared to the PBS control group  against T. spiralis infection [T. spiralis gp43 antigen, which has been proved to be a highly immunodominant protein.During the past decades, researchers have been devoted to developing vaccines against trichinellosis utilizing different vaccine platforms, such as crude worm antigens, recombinant proteins, and DNA vaccines. As aldolase , serine aldolase , DNase Ialdolase , and enoaldolase  from T. hallenge , 26, 27.hallenge . Seven yhallenge . AnotherTs-Pmy has been proved to be a good vaccine candidate antigen which induced significant protection in immunized mice against the challenge of T. spiralis-infective larvae [In our previous study, e larvae . Howevere larvae . In recee larvae . Due to e larvae , 33. SomPlasmodium falciparum  [Yersinia pestis [P. falciparum sporozoites [P. falciparum, anti-MAP-1 (circumsporozoite protein-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites  [However, peptide vaccine also has some disadvantages, such as poor immunogenicity and the need to conjugate to a large carrier protein . To increase the immunogenicity of a peptide-based vaccine, a new strategy of multiple antigenic peptides (MAPs) becomes a popular alternative. MAPs are peptides that are artificially branched by utilizing a lysine-based central backbone to which multiple peptide chains could be conjugated. The branched peptides sometimes can greatly increase their immunological responses because of the high molar ratio of peptide antigen to the core molecule and the high molecular weight which make them more immunogenic without the need of conjugation to a carrier protein , 34–36. ciparum) , Yersinia pestis , and HIVa pestis . Clinicarozoites . In lymprozoites . In P. f BALB/c) . This stTs-Pmy and evaluated its immunogenicity and the induced protective immunity compared to the B cell epitope-conjugated MAP.Among peptide vaccines, epitope-composed one has some other unique advantages and becomes a good choice for vaccine development. This type of vaccine allows us to focus on the epitopes that possess strong immunogenicity and could induce robust protective immune effects in immunized animals. Identification and correct selection of these epitopes are a crucial step in the design of an epitope-based peptide vaccine. The immunogenic epitopes on the protein of interest should be identified either by epitope prediction algorithms using bioinformatics tool  or by scT. spiralis infection [T. spiralis infection. During the initiation of immune response, the major histocompatibility complex- (MHC-) II combined with processed antigen epitope in antigen presenting cells trigger the activation of T helper cells which further activate cellular immunity and/or humoral immunity. In our previous work, based on the BALB/c mouse model, H-2d-restricted CD4+ T cell epitopes (I-Ad and I-Ed) of Ts-Pmy were predicted using the SYFPEITHI database. The epitope peptides could stimulate splenocytes of rTs-Pmy-immunized mice to secrete the Th2 cytokines IL-4 and IL-5. It has been further verified that these epitopes were immunodominant Th2 epitopes of Ts-Pmy by experiments in vitro and in vivo [+ T cell epitopes, stimulation of splenocytes from mice immunized with rTs-Pmy produced the highest IL-5 by peptide T5, while T2 stimulated the highest production of Th2 cytokine IL-4, as compared to the other T cell epitopes. T5 also stimulated splenocytes of mice immunized with T5 to produce the highest IL-4 among all the candidate CD4+ T cell epitopes [+ T cell epitopes to construct MAP in this study. In addition, a protective B epitope of Ts-Pmy, YX1, located between 88 and 107 amino acids of Ts-Pmy, has been identified by the recognition of a mAb 7E2 which passively transferred the protection against T. spiralis infection in naïve mice [T. spiralis infection compared with B cell epitope alone, two types of MAP including MAP-TB and MAP-B were constructed. MAP-TB combines two Th2 epitopes and one B cell epitope while MAP-B contains only B cell epitope  compared to the MAP-B which induced only 12.4% ML reduction. MAP-TB did not induce comparable protection as that induced by rMEP possibly due to the less protective epitopes included in the construct of MAP-TB. Due to technical limitations of MAP synthesis, only two of four CD4+ T cell epitopes and one B cell epitope were constructed in MAPs in this study. The nonsterilizing immunity or low protection is a dilemma not only for vaccine development against Trichinella infection but also for all other helminth infections. For example, one of the most well-researched helminth diseases, schistosomiasis, the bar to achieve protective efficacy in humans was set at a consistent induction of 40% protection or better by the World Health Organization (WHO), and although this is a modest goal, it is yet to be reached with the six most promising schistosomiasis vaccine candidates  [Our previous study showed that a recombinant multiepitope protein (rMEP) combined four CD4eduction . In thisnd Sm23) , 45. Vacnd Sm23) , 47. Thend Sm23) . Indeed,nd Sm23) .γ and IL-2). Th1 cytokine response was also elevated, and it was speculated that the epitopes were short peptides and they might be cross-presented by the dendritic cells.In this study, MAP-TB induced robust humoral immune response with higher IgG titers than MAP-B did. IgG subtype examination showed that MAP-TB immunization induced both IgG1 and IgG2a (with IgG1 predominant), while MAP-B mainly induced IgG1, indicating MAP-TB stimulated both Th1 and Th2 responses. The cytokine profiles secreted by the splenocytes also showed that MAP-TB immunization induced not only Th2 cytokines (IL-4 and IL-5) but also Th1 cytokines  cells are a specialized subset of effector Treg cells that inhibit antibody production . Success+ and CD8+ T cells, and antigen-presenting cells, in vitro and in vivo [Heligmosomoides polygyrus [Schistosoma japonicum [Schistosoma mansoni [Brugia malayi [T. spiralis infection [P = 0.052) and Tfr cells and stimulated Tfh and GC B cells in dLNs and spleens of immunized mice compared to MAP-B alone, indicating the addition of T-epitope in MAP-B could offset the immune inhibition induced by helminth infection and boost humoral immune response to vaccine antigen. This may also partly explain the protective effects induced by the immunization of MAP-TB and MAP-B.Treg cells are considered negative regulators of immune response which could suppress the activation, proliferation, and effector functions (such as cytokines production) of natural killer (NK) and NKT cells, B cells, CD4 in vivo . As a su in vivo . Many heolygyrus , Schistoaponicum , Schisto mansoni , and Brua malayi , are knonfection . In our + T cell epitopes could induce better protection against T. spiralis infection, which was associated with enhanced humoral immune response, compared to the MAP-B and PBS groups. The results from this study indicate that B-epitope alone is not efficient to induce robust humoral immune response. It is necessary to combine CD4+ T cell epitopes to construct an epitope-based peptide vaccine in order to induce better protection against T. spiralis infection. Similar studies on infective diseases also showed that potent CD4+ T cell epitope is a key vaccine component to elicit robust immune responses [T. spiralis. In our future study, including more protective epitopes (both T and B cell epitopes) in MAP constructs would be a practical way to improve protection.In the current study, we found that peptide vaccine MAP-TB comprising CD4esponses , 74. As esponses , therefo


Surfactant protein-D (SP-D), a member of the collectin family has been shown to induce apoptosis in cancer cells. SP-D is composed of an N-terminal collagen-like domain and a calcium-dependent carbohydrate recognition domain (CRD). Recently, we reported that a recombinant fragment of human SP-D (rfhSP-D), composed of homotrimeric CRD region, induced intrinsic apoptotic pathway in prostate cancer cells. Here, we analyzed the membrane interactome of rfhSP-D in an androgen-independent prostate cancer cell line, PC3, by high resolution mass spectrometry and identified 347 proteins. Computational analysis of PPI network of this interactome in the context of prostate cancer metastasis and apoptosis revealed Glucose Regulated Protein of 78 kDa (GRP78) as an important binding partner of rfhSP-D. Docking studies suggested that rfhSP-D (CRD) bound to the substrate-binding domain of glycosylated GRP78. This was further supported by the observations that human recombinant GRP78 interfered with the binding of rfhSP-D to anti-SP-D polyclonal antibodies; GRP78 also significantly inhibited the binding of recombinant full-length human SP-D with a monoclonal antibody specific to the CRD in a dose-dependent manner. We conclude that the interaction with rfhSP-D is likely to interfere with the pro-survival signaling of GRP78. Four SP-D trimeric subunits assemble as a cruciform dodecamer, and their further oligomers appear as fuzzy balls under electron microscope -2, and TLR-4 to bring about immune regulation (Surfactant protein D (SP-D) recognizes an array of carbohydrate moieties present on the microbial surfaces , 2. SP-Dcroscope –5. Pathocroscope . The CRDgulation , 8.via TNF-α/Fas pathway irrespective of the p53 status  and ability to invade  composed of neck and CRD region induced apoptosis in activated eosinophils of allergic patients ; this in3 status . Furthero invade . A signio invade .Reduced expression of SP-D has been observed in the glandular cells of human prostate cancer tissues. SP-D levels negatively correlated with Gleason score, a scoring system based on the cancer cell types and patterns on the tissue sections as well as tumor volume . We receThe broad spectrum anti-cancer activity of rfhSP-D is possibly due to the simultaneous targeting of multiple growth factors, kinases, transcription factors, and apoptotic pathways –13, 16. α), and activating transcription factor 6 (ATF6). However, under ER stress conditions, unfolded proteins titrate away GRP78 to activate the unfolded protein response (UPR) and alleviate cellular stress. Activated UPR decreases protein influx into the ER and induces the synthesis of components involved in protein folding to support cell survival (GRP78 is an endoplasmic reticulum (ER) resident chaperone and is also known as HSPA5. GRP78 acts as an ER stress sensor and is upregulated under stress conditions, for maintaining ER homeostasis and cell survival. For cellular homeostasis, GRP78 interacts with three ER stress sensor proteins: protein kinase RNA-like ER kinase (PERK), inositol-requiring kinase 1  that mimics biochemical changes in advanced prostate cancer, has been significantly responsive to rfhSP-D induced apoptosis  pLysS (Invitrogen), rfhSP-D was expressed and purified as described earlier , followed by the neck region (203–235 aa residues) and eight Gly-X-Y repeats from the collagen domain (179–202 aa residues). Using 6/ml) were pre-incubated with in serum-free RPMI medium containing rfhSP-D (20 µg/ml) and 5 mM CaCl2 for 2 h at 37°C in the CO2 incubator (PC3 cells were pre-incubated with rfhSP-D to enable isolation of complexes of rfhSP-D and membrane proteins). Following incubation, the unbound rfhSP-D was removed along with the medium followed by a wash with sterile PBS. Then, PC3 cells were harvested for membrane protein isolation by scrapping in the wash solution supplied with Mem-PER™ Plus Membrane Protein Extraction Kit .PC3 cells  and incubated overnight at 4°C with mixing. First, Pierce Protein A/G Magnetic beads  were washed three times with TBST wash buffer. The antigen and antibody mixture was added to 1.5 ml tube containing pre-washed protein A/G magnetic beads (250 µg) and incubated at room temperature for 2 h on a rotary shaker. Beads were then separated on a magnetic stand, and the flow-through was saved for analysis. Beads were washed three times with the wash buffer and a final wash was given with Milli Q water. Beads were then resuspended in the elution buffer  and incubated at room temperature for 10 min with mixing. Beads were separated magnetically and supernatant containing the target antigen was saved for further analysis. Low pH was neutralized by adding neutralizing buffer  to the eluate. PC3-derived membrane protein fraction used for LC-MS/MS analysis was pooled from five independent pull-down experiments with three technical replicates.6) in serum-free RPMI medium were plated in a T75 tissue culture flask and pre-incubated with rfhSP-D (20 μg/ml) for 2 h, as described above. PC3-membrane as well as cytoplasmic proteins were isolated, as described above, and analyzed by Western blotting. Lysate proteins (30 μg) were subjected to 12% v/v SDS-PAGE and electro-transferred onto PVDF membranes . To confirm the presence of rfhSP-D binding proteins in the two protein fractions, one of the blot was blocked , incubated with rfhSP-D in the presence of CaCl2 or EDTA (2 h at 37°C), and probed with primary rabbit anti-human SP-D polyclonal conjugate (overnight at 4°C) and secondary goat anti-rabbit Horseradish Peroxidase (HRP) conjugate (1 h at room temperature on a shaker). To confirm the purity of the two protein fractions, another blot was probed with primary antibodies against human Prostate-Specific Membrane Antigen (specific to the membrane protein fraction of prostate cells)  overnight at 4°C, followed by HRP-conjugated secondary goat anti-rabbit IgG antibodies (diluted 1:6000) (1 h at room temperature on a shaker). The blots were developed using the enhanced chemiluminescent (ECL) Kit . The blot images were captured using the Syngene (Chem Genius).PC3 cells  and then subjected to IgG depletion using Multiple Affinity Removal System Spin Cartridge, HSA/IgG , following the manufacturer’s instruction. A buffer exchange followed using Amicon Ultra-0.5 (3K) device in PBS. Total protein was estimated by Bradford assay, followed by SDS-PAGE analysis to assess the profile of cell lysate, and membrane fraction with rfhSP-D treatment after immune-depletion.via 12% v/v SDS-PAGE and electro-transferred to PVDF membranes. Then, the membrane was blocked and incubated with the rabbit anti-human SP-D polyclonal antibodies (overnight at 4°C) and secondary goat anti-rabbit HRP conjugate (1 h at room temperature). The blot was developed using the enhanced chemiluminescent (ECL) Kit . Image was acquired by Syngene (Chem Genius).To confirm the presence of rfhSP-D in the two protein fractions (20 μg), the supernatant of pull-down fraction (5 μg), flow-through of pull-down fraction (5 μg), eluate of pull-down (5 μg), and flow-through containing IgG after immune-depletion (5 μg) were separated via C-18 columns and eluted with a buffer comprising acetonitrile (40%) and formic acid (0.1%). Fractionated peptides were lyophilized on a SpeedVac (30 min at 45°C) and then solubilized in 0.1% formic acid prior to LC-MS/MS analysis.Eluates of the five individual pull-down experiments were diluted in ammonium bicarbonate buffer  to a final volume of 100 μl, followed by reduction using 10 mM dithiothreitol (DTT) for 20 min at 60°C. Once the proteins came to room temperature, they were alkylated  for 10–15 min at room temperature in the dark. The proteins were digested with 1 μg of Lys-C for 3–4 h at room temperature, followed by digestion with 2 μg of trypsin overnight at 37°C. Enzyme activity was terminated with 1% v/v formic acid. Peptides were fractionated 5 for MS and 1 × 105 for MS/MS.Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific), connected with an Easy-nLC II nanoflow liquid chromatography system (Thermo Fisher Scientific), was used for peptide analysis. A trap column  was used to enrich peptides. The peptides were separated at a flow rate of 30 ml/min on a 20 cm long column of 5 µm Magic-C18-AQ  using a gradient of 8–30% solvent B (90% acetonitrile in 0.1% formic acid) over 103 min for a 120 min run. Mass spectrometry data were collected at a resolution of 120,000 in a range of 350–1,600 m/z. The highly intense ions with charge state >2 were isolated in 3 s cycle and subjected to HCD fragmentation with 32% normalized collision energy. These fragmented ions were sensed at a resolution of 15,000 at 200 m/z. The limit of dynamic exclusion was fixed at 40 s with a 10-ppm mass window. The maximum ion injection times were 50 ms for MS and 75 ms for MS/MS. The automatic gain control targets were 4 × 10http://www.proteomexchange.org) via the PRIDE partner repository (dataset identifier PXD008098).The uninterpreted MS/MS data from the complete LC-MS/MS run was subjected to a search in the database (Human RefSeq protein database). The search algorithm used was SEQUEST, and the platform used was Proteome Discoverer . A maximum of two missed cleavages, carbamidomethylation at cysteine as fixed and oxidation of methionine as variable modifications, were included as the search parameters. Mass tolerance of monoisotopic peptide was set at 10 ppm and the MS/MS tolerance was limited to 0.02 Da. At the PSM as well as the protein level, the false discovery rate of 1% was set. MS data has been submitted to the ProteomeXchange Consortium (http://www.ingenuity.com)  software (ity.com) .Protein–protein interactions (PPIs) of the rfhSP-D interactome (347 proteins) were downloaded from STRING v11 . ProteinEscherichia coli BL21) (PDB ID: 5E84 Chain A) was downloaded from PDB  since the O-linked glycosylated GRP78 specific to cancer cells is a minor fraction of the total GRP78 . Bound rfhSP-D was probed with primary rabbit anti-human SP-D polyclonal antibodies (1 h at 37°C) and secondary goat anti-rabbit HRP conjugate (1 h at 37°C), or with biotinylated Human SP-D detection antibody  (1 h at room temperature with shaking) and Streptavidin-HRP conjugate .To analyze the rfhSP-D/rFLhSP-D  interaction with GRP78, the study utilized non-glycosylated version of GRP78  in duplicates, followed by blocking and incubation with GRP78 (1 µg/ml in PBS). The wells were then probed with primary rabbit anti-human SP-D polyclonal antibodies and secondary goat anti-rabbit HRP conjugate.−3 nM) and increasing concentrations of human recombinant GRP78 from 5 ng/ml (70 × 10−3 nM) up to 20 ng/ml (280 × 10−3 nM) (1 h at 37°C), and probed with Human SP-D detection antibody (500 ng/ml) (1 h at room temperature with shaking). After washing, the wells were incubated with Streptavidin-HRP conjugate (1:200) (45 min at room temperature). Color was developed using a substrate solution  was analyzed by ingenuity pathway analysis (IPA) for associated biological functions, canonical pathways, and networks. Our network analysis revealed novel signaling proteins that are likely to interact (directly/indirectly) with rfhSP-D and/or regulate its associated networks  element (−109) wherein transcription factors of the ies bind . Foxm1 ries bind . CCAAT-e41 cells .Materials and Methods) to shortlist critical proteins of the rfhSP-D interactome. 20 proteins  were selected and ranked based on connectivity, perturbation, and disruption scores  from blind docking of rfhSP-D and GRP78 revealed that CRD of SP-D can bind with GRP78 via the substrate binding domain of GRP78  and SAAENAALQQLVVAK (positions 293–307) that are located in the CRD region of SP-D  was observed due to the blocking/masking of antibody-recognized domains by GRP78 (1 µg/ml i.e. 0.280 nM).To evaluate if GRP78 binding can interfere with the interaction of rfhSP-D and polyclonal antibodies, a direct ELISA was carried out using rfhSP-D coated on the microtitre wells, followed by GRP78 as the ligand and probed with polyclonal antibodies. Minimal binding of polyclonal anti-human SP-D antibody to rfhSP-D  using the monoclonal anti-human SP-D antibody. The assay detected rFLhSP-D from 0.115 to 10 ng/ml in a linear standard curve (i.e. 281 × 10−3 nM) on binding to rfhSP-D (5 ng/ml i.e. 116 × 10−3 nM) showed approximately 57.15% inhibition of binding to monoclonal anti-human SP-D antibodies. Thus, GRP78 interfered with the interaction between rFLhSP-D and monoclonal anti-human SP-D antibody, suggesting that CRD of rFLhSP-D is likely to be involved in its binding to GRP78.To examine if GRP78 binding to rFLhSP-D (recombinant full-length human SP-D) is in silico and in vitro experimental analysis.To delineate the underlying mechanisms of the anti-prostate cancer activity of rfhSP-D, we used a pull-down strategy to unravel the rfhSP-D interactome from the membrane fraction of the metastatic prostate cancer cell line, PC3. A high-throughput proteomic work flow led to the identification of 347 membrane proteins with ≥2 unique peptides, each with ≥2 PSMs. Based on the PPi network properties such as connectivity, perturbation, and disruption scores, 20 proteins were selected, including GRP78 that probably interacted directly or indirectly with rfhSP-D. The interaction between rfhSP-D and GRP78 was validated by We recently reported a differentially increased binding of rfhSP-D to the metastatic prostate cancer cells and induction of the apoptosis . The memvia appropriate protein folding, inhibiting aggregation of newly-made proteins, and regulating the stimulation of transmembrane sensors in the endoplasmic reticulum (ER) , is a member of the HSP70 superfamily, and has a critical role in the regulation of the unfolded protein response (UPR) lum (ER) . Expresslum (ER) . Prostatlum (ER) . Environlum (ER) –60.GRP78 has been primarily localized to the ER owing to the presence of the KDEL-retention motif, although a minor proportion of GRP78 evades the ER-retention mechanism and reaches the cell surface to promote cell survival . Proteomα2-macroglobulin (α2-M), tumor differentiation factor, and vaspin via its substrate binding domain and induce AKT/PI3K pro-survival pathway  interaction, causing downregulation of the EGF induced signaling in A549 cell line  can be found in the article/in silico molecular docking studies. PG and BB performed and analyzed LC-MS/MS data by IPA software. SI-T, BB, PG, RS, and UK reviewed the first draft and provided valuable suggestions. TM defended the proposal to acquire the Institutional grant and IEC approval, analyzed the data, and edited the manuscript. All authors contributed to the article and approved the submitted version.Conception, co-ordination, and design of the study were accomplished by GT and TM. GT performed and analyzed the experiments and prepared the first draft of the manuscript. SA and UK cloned, recombinantly expressed, and characterized the purified rfhSP-D used in the study. RS, PG, and GS designed, performed, and analyzed the LC-MS/MS data. SI-T and IK designed, executed, and analyzed network and Intra-mural grant allocated to TM by the Institute ICMR-NIRRH (Accession no. 921) was used for all the consumables used for experiments. ICMR-NIRRH-JRF and ICMR-SRF supported GT’s salary.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Data of molecular dynamics (MD) simulations were obtained for mucosal-associated invariant T (MAIT) cell ligands complexed with MR1 or MR1/TCR. Ligands included in the simulations were natural ligands 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), 5-(2-oxopropylideneamino)-6-(D-ribitylamino)uracil (5-OP-RU), their C5’ ethinylated analogs in S or R configuration, as well as the corresponding fluorophore-reacted products. All-atom models of the binary and ternary complexes were constructed using PDB entry 4NQE and docked poses  The data permit a MD weighted appreciation of crystallographically observed interactions. Thus, they may support drug design of MAIT cell ligands. They were notably generated from models of both, binary and ternary complexes of MAIT cell ligands to give insights into the formation of the ternary complexes.•Researchers or scientists i) working in the field of MAIT cell biology, specialized in structural biology or molecular modelling or particularly interested in MD of ternary ligand/MR1/TCR or binary ligand/MR1 complexes or ii) interested in fluorophore/protein interactions may benefit from access to these data.•Trajectories can be visualized for appreciation of the molecular dynamics of both, ligands and proteins and their interactions; they can be further used for additional analysis of e.g. conformational changes or interactions not explicitly reported in the primary citation. The MD files can also serve to start extensions of the simulations or as input for more sophisticated MD approaches to support drug design.•TM 488 fluorophore-labelled ligand/MR1 complexes as PET requires at least transient van der Waals contacts of excited donor and suitable acceptor groups which can be assessed by MD simulations The data provide a first step to the risk assessment of photoinduced electron transfer (PET) in Alexa Fluor1The MD raw data are deposited in Mendeley Data as tar archives. All input and output files required and created by program Desmond as commercialized by Schrodinger Inc. are provided. All files were compressed prior to archiving. The trajectory files found in the directories [MDsimulation_name]_trj have to be decompressed prior to visualization. The 4 simulations of the binary and ternary complexes of 5-OE-RU and 5-OP-RU are found in archive_natural_ligands_5OERU.tar and archive_natural_ligands_5OPRU.tar, respectively. The 4 simulations of the ternary complexes of the R and S stereoisomers of their ethinylated analogues are contained in archive_ethinylated_ligands_SSRR_ternary_2.tar and archive_ethinylated_ligands_SSRS_ternary_2.tar, respectively, while the simulations of the corresponding binary complexes are in archive_ethinylated_ligands_binary.tar. The 4 simulations of the binary complexes of the two stereoisomers of the fluorophore-reacted products are accessible in archive_fluorescent_ligands.tar.SIDR.pdf contains the Simulation Interactions Diagram Reports in pdf format of all 16 simulations, as well as the plots of radial distribution functions when relevant. The Simulation Interactions Diagram Reports cover analysis of protein-ligand rmsd, protein rmsf, protein secondary structure, ligand rmsf, protein-ligand contacts, ligand torsion profile, and ligand properties.Statistics_ncic.xlsx reports more detailed statistics of non-covalent interaction counts in xlsx format of all 16 MD simulations.2MD were simulated for ternary and binary ligand complexes of TCR/MR1 and MR1. Starting models were prepared for the natural ligands 5-OE-RU, 5-OP-RU, their ethinylated analogues in R and S stereochemistry and the corresponding fluorophore-reacted analogues, the latter as binary complexes only. The experimental work accompanied by these simulations only used ligand activation by methylglyoxal Molecular modelling was carried out using Drug Discovery Suite by Schrödinger Inc. in versions 2016.u3 or 2020.u1 to visualize models, design and prepare ligands and protein structures, complete protein models by homology modelling, refine loop conformations, minimize energy, simulate and analyze MD. Thus, programs and tools Maestro, Protein Preparation Wizard, Ligprep, Epik, Glide, PRIME, Macromodel, and Desmond were employed by using standard procedures and settings unless specified otherwise 13) were prepared for molecular dynamics simulations by replacing the original ligand in prepared pdb entry 4NQE A assuming ligand activation by either glyoxal or methylglyoxal, and deletion of water molecules sterically interfering with the ligands. Covalent ternary TCR/MR1 complexes of 13 reacted with 3,6-Diamino-9-[2-carboxy-5-carboxamido-(6-azidohexanyl)-phenyl]-4,5-disulfoxanthylium (Alexa TM® 488) fluorophore were prepared from 13 in the docked conformations in the same manner. More precisely, the ethinyl groups were accordingly modified, the fluorophore extensions manually fit to the protein, the covalent linkages constructed, and sterically interfering water molecules deleted. Complexes of 5-OE-RU and 5-OP-RU were prepared from 4NQE. All models were subjected to hydrogen bond optimization and energy minimization of hydrogen atoms in the protein preparation wizard before energy minimization to convergence in Macromodel with a fully flexible zone within 4.5 Å of the ligand, a buffer zone of additional 10 Å, and all other residues frozen while the fillres option was applied. As all published crystallographic models of MR1/TCR complexes lack a few N- or C-terminal residues as well as several short loops, we de novo modelled residues N1H – G3H, C-termini V269A – P270A, D96B – M99B, P198G – S203G, E239H – D245H, loops L246A – L253A, R122G – S130G, N176G – D179G, and F201H – R210H. Thus, homology models of chains A, B, G, and H were built on the energy optimized models using Prime to include missing residues. The inserted residues were subjected to sequential, extended loop refinement in Prime. These models replaced the original chains; chains G and H were omitted in models for simulations of ligand/MR1 binary complexes. Model completion issued a variety of alternative conformations for the missing residues of the 20 completed complexes  for 120 ns production phase at 300 K and 1 atm applying Nose-Hoover chain thermostat and Martyna-Tobias-Klein barostat methods. Atomic coordinates (frames) were saved every 60 ps, energy values logged every 12 ps. The production phase was preceded by the standard preequilibration protocol provided with Desmond. They were further analysed by event_analysis.py and analyze_simulation.py scripts provided by Schrödinger. Standard quality and structural analysis showed stable simulations in terms of potential and total energy, volume, temperature, pressure, and secondary structure (SIDR.pdf). In none of the simulations rmsf of the protein chains exceeded 7.0 Å, thus illustrating moderate conformational changes (SIDR.pdf). Rmsf of de novo modelled loops were observed at the same level as crystallographically observed loops. Detailed analysis and precise values of non-covalent intraligand and ligand-protein interactions are reported in supplementary materials SM2 of Data generation, acquisition, and analyses did not involve experimentation with human subjects or animals. Neither were data collected from social media platforms.Anke Steinmetz: Conceptualization, molecular modelling, MD simulations and their analyses, Knime script, Writing - Original draft preparation, editing, finalization; Thomas Yvorra, Pascal Retailleau, Olivier Lantz, Frédéric Schmidt: Writing – Reviewing & co-submission.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article. Else, Anke Steinmetz is an employee of Sanofi-Aventis R&D.


ADT-A and ADT-D, were enhanced in compression wood of maritime pine, a xylem tissue enriched in lignin. Transcriptomic analysis of maritime pine silenced for PpMYB8 revealed that this gene plays a critical role in coordinating the deposition of lignin with the biosynthesis of phenylalanine. Specifically, it was found that ADT-A and ADT-D were strongly down-regulated in PpMYB8-silenced plants and that they were transcriptionally regulated through direct interaction of this transcription factor with regulatory elements present in their promoters. Another transcription factor, PpHY5, exhibited an expression profile opposite to that of PpMYB8 and also interacted with specific regulatory elements of ADT-A and ADT-D genes, suggesting that it is involved in transcriptional regulation of phenylalanine biosynthesis. Taken together, our results reveal that PpMYB8 and PpHY5 are involved in the control of phenylalanine formation and its metabolic channeling for lignin biosynthesis and deposition during wood formation in maritime pine.Biogenesis of the secondary cell wall in trees involves the massive biosynthesis of the phenylalanine-derived polymer lignin. Arogenate dehydratase (ADT) catalyzes the last, and rate-limiting, step of the main pathway for phenylalanine biosynthesis. In this study, we found that transcript levels for several members of the large ADT gene family, including  Functional genomics unravels the role of two arogenate dehydratases in maritime pine during wood formation, and provides new insights into how lignin biosynthesis and deposition are regulated in conifers. The biosynthesis of the amino acid phenylalanine (Phe) is a doubly essential process for land plants, as this amino acid serves both as a building block for proteins and as a main precursor for the biosynthesis of phenylpropanoids. Phenylpropanoids are a wide range of aromatic compounds that play key roles in plant growth, development, and the response to environmental cues. Land colonization by the first terrestrial plants would not have been possible without the emergence of specialized metabolic pathways for the biosynthesis of these secondary metabolites .enolpyruvate (PEP) and erythrose-4-phosphate (E4P), which are ultimately converted into chorismate, the direct precursor for the biosynthesis of aromatic amino acids and others compounds, such as vitamins K1 and B9 , a specialized vascular tissue with an enhanced deposition of lignin  that belongs to the bZIP family of TFs, is also able to bind to the regulatory regions of the PpADT-A and PpADT-D genes. Interestingly, the gene expression pattern of PpHY5 is opposite to that of PpMYB8 in CW, suggesting an opposite role for these two genes in lignification.Despite the fact that they can act as a bottleneck in Phe biosynthesis  vector. Transformation was performed by co-cultivation of embryonal suspensor masses with Agrobacterium tumefaciens carrying the corresponding OE or RNAi vectors , and confirmed by sequencing.airs see : p1830.5D0 parameter) for each gene was deduced from the Mass Action Kinetic 2 model using the qPCR package for the R environment  was used. Slides were made by Agilent Technologies and hybridization was performed as described by The 60-mer oligonucleotides custom microarray PINARRAY3 (Escherichia coli BL21-AI™ (Thermo Fisher Scientific) by overnight culture at 12 °C. The oligonucleotide probes described in The recombinant protein PpMYB8 (FN868598) was produced in PpADT-A were amplified from the pJET1.2 construct using the reverse primer PAAttB1R paired with three different forward primers: PA1AttB4 (638), PA2AttB4 (394), and PA3AttB4 (246). The corresponding PCR products were re-amplified using the primers AttB4 and AttB1R, and cloned into Gateway® pDONR-P4-P1R (Thermo Fisher Scientific) vector using BP Clonase® II, and subsequently recombined into pMW#3 , and PDAttB1R (reverse). PpADT-D promoter deletions were subcloned as described for PpADT-A. All primers used for promoter analysis in yeast are listed in Deletions from the 5' end of PpMYB8 prey construct was done by amplifying the PpMYB8 ORF from a construct previously available in our laboratory  as substrate. More details can be found in Supplementary Protocols S1.Stems previously preserved in liquid nitrogen were fixed overnight in a 4% paraformaldehyde solution in phosphate-buffered saline (PBS) and included in paraffin. Transversal sections (10 µm) were deparaffinized and rehydrated before visualization. For Wiesner (phloroglucinol-HCl) staining, a few drops of phloroglucinol-HCl solution (two parts of 1% phloroglucinol in 95% ethanol and one part of pure HCl) were deposited over the tissue sections and observed immediately under white light. For lignin autofluorescence visualization, an excitation range of 460–500 nm and an emission range of >510 nm were used. In both cases (Wiesner staining and lignin autofluorescence), a Leica TL3000 Ergo stereo-microscope was used to visualize and register the results.Acid-soluble and -insoluble lignin were determined in cell wall preparations from ~500 mg of fresh ground stems preserved at –80 °C. Acid-insoluble lignin was determined from ~100 mg of cell wall preparation using the Klason method  with theP. pinaster ADT gene family, consisting of nine identified isoforms , prephenate aminotransferase (PpPAT), phenylalanine hydroxylase (PpPH), and phenylalanine ammonia lyase PpPAL1  from ~1.5 to 6; PpMYB8 overexpression was observed in roots where paradoxically a certain degree of silencing was perceptible, particularly in the line 12PP22.Compared with controls (RNAi-EV), RNAi- 2 years . In conteenhouse . EndogenPpMYB8 regulates the expression of ADT genes during xylogenesis, the expression levels of the ADT genes were determined in the stems of the RNAi-PpMYB8 lines  was found to be overexpressed , which reveals the presence of lignin in a quantitative manner, and lignin autofluorescence after excitation under blue light showed that the RNAi-PpMYB8 plants were strongly affected in their xylematic tissues  lignin contents were estimated in the crude cell wall residue from stems of RNAi-PpMYB8 plantlets. The silenced lines exhibited an ~30% decrease in the total lignin content (acid-soluble+acid-insoluble lignin) when compared with the control plants  was searched to identify the 5'-flanking regions of the ADT genes and used as a template to identify and assemble their P. pinaster relatives (SustainPineDB: http://www.scbi.uma.es/sustainpine/)  . The lenPpADT-A and PpADT-D (PpADT-A (PADT-A) analyzed contains 1907 bp upstream of the predicted translation start codon, presenting three AC elements in this region at positions 301 (AC-III element), 247 (AC-II element), and 209 (tandem repeated AC-II element) (see PpADT-D (PADT-D) comprises 1226 bp from the predicted translation start codon, with three AC-II class elements in its proximal region at positions 298, 283, and 206.A close-up analysis of these 5'-flanking sequences showed a particular enrichment of putative R2R3-MYB-binding sites in the proximal region of  PpADT-D . The 5'-cis-AC elements found in the 5'-upstream regions are important for PpMYB8 recognition, we used an EMSA to establish a physical link between PpMYB8 and synthetic DNA probes from PADT-A and PADT-D. The following probes were designed as follows: for PADT-A, probe A1 from position 225 to 329 (105 bp) and probe A2 from 281 to 370 (90 bp); for PADT-D, probe D1 from 253 to 331 (66 bp) and probe D2 from 192 to 318 (127 bp) (To investigate whether the putative (127 bp) . Band sh(127 bp) . The banin vivo direct interaction between PpMYB8 and the putative regulatory regions PADT-A and PADT-D, a β-galactosidase reporter assay was performed in the yeast S. cerevisiae. For PADT-A the following 5'-sequential deletions were assayed: 638 (PA.1), 394 (PA.2), and 246 (PA.3) , PD.2 (331), and PD.3 (205). Similarly, as found for PADT-A, the AC-II-binding elements located at positions 298, 283, and 206 of the 5'-flanking region of PADT-D were found to be critical for the recognition by PpMYB8, as demonstrated by the drastic reduction of β-galactosidase activity in PD.3 (To confirm an 6 (PA.3) . A signi in PD.3 .PpMYB8 plants. A 60-mer oligonucleotide microarray (PINARRAY3) developed in our laboratory and manufactured by Agilent . Among these genes, 307 were up-regulated in plants silenced for PpMYB8 as compared with controls, whereas 477 were down-regulated (sp_v3.0_unigene19258 and sp_v3.0_unigene3263), a bZIP (sp_v3.0_unigene8197), a RAV/NGATHA (sp_v3.0_unigene34155), a MADS box (sp_v3.0_unigene32645), a C3H zinc finger (sp_v3.0_unigene17894), a GARP (sp_v3.0_unigene19789), and a bHLH (sp_v3.0_unigene29985). Consistent with the strong effect on xylem tissue in plants silenced for PpMYB8, we detected down-regulation of genes directly involved in cell wall modification such as xyloglucan endotransglucosylase/hydrolase (sp_v3.0_unigene35083), a wall polysaccharide-specific O-acetyltransferase , a pectinesterase (sp_v3.0_unigene9201), an oxidoreductase (sp_v3.0_unigene2867), a chitinase (sp_v3.0_unigene29478), and up to three S-adenosyl-l-methionine-dependent methyltransferases putatively involved in cell wall biogenesis . Also consistent with the observed phenotype, multiple genes with a role in plant development were down-regulated, including NO VEIN (sp_v3.0_unigene63788), the actin cytoskeleton organizer PIROGI (sp_v3.0_unigene13374), and the mRNA decapping DCP2 (sp_v3.0_unigene183375) whose silencing in Arabidopsis results in disorganized veins. Additionally, an important group of genes involved in cell organization and division were also down-regulated including PROHIBITIN 3 (sp_v3.0_unigene24132), CRUMPLED LEAF (sp_v3.0_unigene1324), and multiple cytoskeleton-related genes   .sp_v3.0_unigene133569) orthologous to the Arabidopsis AtMYB35, DEFECTIVE IN MERISTEM DEVELOPMENT AND FUNCTION 1, a TCP putatively orthologous to the Arabidopsis PLASTID TRANSCRIPTION FACTOR 1 (PTF1) involved in chloroplast regulation of leaf differentiation (sp_v3.0_unigene6586), and a HIGH MOBILITY GROUP (HMG) box protein (sp_v3.0_unigene24902). Interestingly, we also detected up-regulation of tryptophan synthase (sp_v3.0_unigene133330) and enoyl-CoA hydratase/isomerase D (sp_v3.0_unigene22584), key enzymes involved, respectively, in the biosynthesis of Trp and vitamin K1, through two alternative pathways that, as with the Phe/Tyr pathway, start from chorismate. Also related to amino acids, two additional up-regulated genes were detected, an ACT domain repeat protein (sp_v3.0_unigene8593), involved in feedback regulation of amino acid metabolism, and ornithine delta-aminotransferase (sp_v3.0_unigene5775), involved in arginine catabolism. The microarray results were validated by RT–qPCR analysis of a set of DEGs , a pine ortholog of Arabidopsis HY5 (ELONGATED HYPOCOTYL 5), was significantly up-regulated  while, in the 11PP17 line, the adjusted P-value was 0.09. HY5 is a central TF that promotes, among other processes, the accumulation of Phe-derived compounds and the repression of critical genes for cell wall biogenesis. Moreover, there is evidence demonstrating the coordination of HY5 and MYB TFs in different processes related to the synthesis of Phe-derived compounds  and reduced levels of cellulose , prephenate aminotransferase (PpPAT), phenylalanine hydroxylase (PpPH), and phenylalanine ammonia lyase (PpPAL1), were up-regulated in CW compared with OW  of DEGs compared with the controls that were shared by both the 11PP16 and 11PP17 RNAi-PpMYB8 lines, of which 307 genes were up-regulated and 477 were down-regulated , and the AtACR11 protein activates GS2 activity , which is essential for auxin biosynthesis, and thus this supports the possible involvement of auxin in the RNAi-PpMYB8-mediated phenotype.The observed up-regulation of an ACT domain repeat (ACR) protein is particularly interesting since these proteins are involved in feedback regulation of amino acid metabolism and function in modulating the activity of amino acid biosynthetic enzymes .Table S1. Estimated H- and G-lignin content in RNAi-Table S2. Primer sequences used for cloning.Table S3. Primer pairs used for RT-qPCR.PpMYB8 (RNAi- PpMYB8).Table S4. Transcriptome analysis of transgenic plants silenced for PpMYB8 (OE-PpMYB8).Table S5. Transcriptome analysis of transgenic plants overexpressing P. pinaster in the OE-PpMYB8 lines, expressed as a percentage of controlsFig. S1. Average expression level of the nine ADT genes from In silico analysis of candidate AC- and bZIP-binding elements within the 5'-flanking region of the ADT genes in Pinus pinaster.Fig. S2. PADT-A and PADT-D sequences.Fig. S3. Probes designed for the EMSA in the Protocols S1.eraa099_suppl_Supplementary_table_S1-S3_figure_S1-S3Click here for additional data file.eraa099_suppl_Supplementary_data_1Click here for additional data file.eraa099_suppl_Supplementary_data_2Click here for additional data file.eraa099_suppl_Supplementary_protocolsClick here for additional data file.