Arabidopsis thaliana LEC2 (AtLEC2) driven by glucocorticoid receptor-dexamethasone (GR-DEX) inducible system (AtLEC2-GR) triggers embryogenic callus formation in tobacco (Nicotiana tabacum).LEAFY COTYLEDON 2 (LEC2) acts throughout embryo morphogenesis and maturation phase to maintain embryogenic identity. Our previous study stated that AtIPT3, AtIPT7 and the tRNA isopentenyltransferase gene AtIPT9 were overexpressed in the AtLEC2-GR transgenic background. In the AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR seedlings, high-quality embryogenic callus was obtained under the DEX condition, and the shoot regeneration efficiency was 2 to 3.5 folds higher than AtLEC2-GR alone on hormone free medium without DEX. Transcriptome analyses showed that up-regulated BBM, L1L, ABI3, and FUS3 might function during embryogenic callus formation. However, at the shoot regeneration stage, BBM, L1L, ABI3, and FUS3 were down-regulated and Type-B ARRs were up-regulated, which might contribute to the increased shoot regeneration rate.In this study, the adenosine phosphate isopentenyltransferase genes AtLEC2 triggers embryogenic callus formation and overexpression of AtIPT7 or AtIPT9 improves shoot regeneration without exogenous cytokinin.A novel system for inducing shoot regeneration in tobacco has been developed using the GR-DEX system. Induced expression of The online version of this article (10.1186/s12870-019-1907-7) contains supplementary material, which is available to authorized users. Genetic improvement through the transgenic technology has been widely used for many crops, however, the transformation and regeneration of some crops were proved to be difficult and genotype dependent . EstabliLEAFY COTYLEDON 1 (LEC1) [LEAFY COTYLEDON 2 (LEC2) [WUSCHEL (WUS) [BABY BOOM (BBM) [AGAMOUS-LIKE 15 (AGL15) [LEC1/LEC1-LIKE (L1L) with three B3 domain protein genes ABSCISIC ACID (ABA)-INSENSITIVE3 (ABI3), FUSCUA3(FUS3) and LEC2 is referred as LAFL network  [FUS3 and ABI3 enhances accumulation of embryo traits but without somatic embryogenesis [YUCCA4 (YUC4) encodes a protein that catalyzes the rate-limiting step in IAA biosynthesis [YUC4 promoter [LEC2 rapidly activates YUC2 and YUC4 [AGL15 [gibberellin 20-oxidase1 (GA20ox1), GA3ox1 and GA3ox2 GAs biosynthesis enzyme encoding genes [. Therefore, the embryogenic competence of LEC2 is also associated with GAs activity. In addition, the expression of LAFL genes is regulated at both chromatin level and transcriptional level. Two AINTEGUMENTA-LIKE (AIL) family transcriptional factors, BBM and PLETHORA2 (PLT2), may directly activate LAFL transcription [LAFL gene expression [LEC2 transcription [Transcriptional factors are known to play significant roles in plant cell differentiation and dedifferentiation. Overexpression of a number of transcriptional factor genes can improve somatic embryogenesis and enhance plant regeneration, such as 1 (LEC1) , LEAFY C2 (LEC2) , 6, WUSCEL (WUS) , BABY BOOM (BBM)  and AGAM AGL15) . LEC1/LEnd LEC2) . This nend LEC2) . Ectopicogenesis , 13. YUCynthesis . LEC2 ca . LEC1/Land YUC4 . LEC2 di4 [AGL15 , 17. AGLng genes . Therefocription . VIVIPARpression , CURLY Lcription , 24.AtLEC2 by the GR-DEX system (AtLEC2-GR) display an obvious somatic embryogenesis phenotype and shoots could be generated from the embryogenic callus under no DEX condition [LEC2 can trigger vegetative to embryogenic transition, however, plant regeneration could not occur from embryogenic callus constitutively expressing LEC2 , 6. The Dictyostelium discoideum and Agrobacterium tumefaciens, it has been proved to convert adenosine-5′-monophosphate (AMP) and dimethylallyl pyrophosphate (DMAPP) into isopentenyladenine riboside 5′ -monophosphate (iPMP) [IPT from the Ti-plasmid of A. tumefaciens significantly elevates the cytokinin levels in transgenic plants and results in excessive cytokinin abnormal phenotype [IPT from the Agrobacterium tumefaciens by the GR-DEX system can induce suitably elevated cytokinin levels and shoot formation [AtIPTs show diverse temporal and spatial expression patterns [AtIPT4 enhanced shoot regeneration efficiency independent of external cytokinins [AtIPT8 gain-of-function mutant remarkably increases iPMP and Isopentenyl adenosine (iPA) levels, improves embryogenic callus formation and shoot regeneration [Arabidopsis thaliana histidine phosphotransfer proteins, AHPs), His kinases (HKs) and Arabidopsis response regulators (ARRs) [ARRs  can activate the expression of WUSCHEL (WUS), to maintain the shoot apical meristem and axillary meristem. Furthermore, these proteins can negatively regulate YUCCAs (YUC1 and YUC4) to inhibit auxin accumulation [yuc1 yuc4 double mutant is dramatically decreased, suggesting that auxin is also indispensable in the determination of cell fate [Cytokinins control cell division and cell differentiation, as well as shoot growth and apical dominance. It is well-known that the appropriate ratio of cytokinin and auxin promotes shoot formation . Isopente (iPMP) , 31. Conhenotype . Howeverormation . In Arabormation . AtIPTs patterns –36. Overtokinins . Furtherneration . The cyts (ARRs) . These fs (ARRs) –42. Recemulation –45. Howeell fate .LEC2, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERKs), WUS and BBM [BBM and WUS2 genes significantly increases transformation frequency in commercial maize inbred lines, sorghum, sugarcane, and indica rice. Here, we described a simple and efficient regeneration system that combines the use of AtLEC2-GR and AtIPT7 or AtIPT9 overexpression. The system relied on the DEX-inducible expression of AtLEC2 to trigger embryogenic callus formation and overexpression of AtIPT7 or AtIPT9 to promote shoot regeneration. This regeneration system enabled embryogenic callus formation under the DEX condition and shoot regeneration after the removal of DEX.High regeneration and transformation efficiency are crucial for gene engineering-based crop genetic modification. Many studies have attempted to increase somatic embryogenesis and shoot regeneration rate through ectopic expressing key genes, such as  and BBM , 46–50. AtLEC2 under the DEX condition can generate embryogenic callus on the shoot apical meristem (SAM) in transgenic tobacco [AtLEC2-GR transgenic line.Induced expression of  tobacco . Shoots  tobacco . In ordeAtLEC2-GR embryogenic callus through increasing endogenous cytokinin concentration. IPTs are key genes in the cytokinin biosynthesis pathway. AtIPT3, AtIPT7 and AtIPT9 were proved mainly expressed in phloem tissues or proliferating tissue of the growing seedling [AtIPT3、AtIPT7 and AtIPT9 overexpressed vectors driven by the CaMV 35S promoter were constructed and then transformed into the AtLEC2-GR transgenic tobacco  respectively by the Agrobacterium-mediated leaf disc transformation [AtIPT3-OE AtLEC2-GR, 15 AtIPT7-OE AtLEC2-GR and 18 AtIPT9-OE AtLEC2-GR independent transgenic lines were generated, respectively. As shown in Additional file AtLEC2, AtIPT3, AtIPT7 and AtIPT9 fragments were amplified by PCR using specific primers in the indicated transgenic lines. The result suggested that AtIPT3, AtIPT7 and AtIPT9 had been stably integrated into the genomes of AtLEC2-GR transgenic tobacco. In the subsequent experiment, three independent AtIPTs-OE AtLEC2-GR transgenic lines of each construct were selected for further studies.The prominent effect of exogenous cytokinin treatment encouraged us to promote the shoot regeneration efficiency from the seedling . AtIPT3、ormation . TotallyAtIPTs-OE AtLEC2-GR transgenic seeds were germinated on MS medium with or without the DEX induction. The comparisons of the AtIPTs-OE AtLEC2-GR lines with wild type and AtLEC2-GR lines were carried out. Under no DEX condition, no remarkable phenotype difference was observed among the lines of AtIPTs-OE AtLEC2-GR, AtLEC2-GR and wild type plants , on the contrary, the primary root length of AtIPTs-OE AtLEC2-GR was 22.3, 34.6 and 16.9% shorter than the control seedlings, respectively  -type cytokinin contents in the 20 DAG AtIPTs-OE AtLEC2-GR, AtLEC2-GR and wild type seedlings grown on MS medium with 20 μM DEX, but no significant change was observed (data not shown).When grown on MS medium with 20 μM DEX, we could not observe embryogenic callus formation on the SAM in all 13 AtIPT7-OE AtLEC2-GR, AtIPT9-OE AtLEC2-GR and AtLEC2-GR under the 20 μM DEX induction were cultured on the hormone-free MS medium without DEX  plants, 6310 genes were up-regulated and 10,789 genes were down-regulated  protein genes were significantly upregulated. The expression of SUCROSE SYNTHASE 2 (SUS2) and FATTY ACID DESATURASE 2 (FAD2) genes were upregulated  were significantly up-regulated in the AtLEC2-GR seedlings  auxin efflux facilitators  and auxin response factors (ARFs)  , VP1/ABI3-LIKE 1 (VAL1) and CHD3-type chromatin-remodeling factor PICKLE RELATED 2 (PKR2), were up-regulated   . GO terms of response to stimulus (GO:0050896), response to stress (GO:0006950) and so on, were enriched in the differentially expressed genes  plants , 2846 genes were up-regulated and 8006 genes were down-regulated . GO analysis of differentially expressed genes revealed enrichment in carbohydrate metabolic process (GO:0005975), generation of precursor metabolites and energy (GO:0006091) and response to stimulus (GO:0050896)  , YUC4, ARF5, ARF8 and GA20ox2 were expressed oppositely when compared with AtLEC2-GR and two other AtIPTs-OE AtLEC2-GR plants . Carbohydrate catabolic process (GO:0016052), carbon fixation (GO:0015977) and hexose metabolic process (GO:0019318) and so on, were enriched in the differentially expressed genes   were used for gene expression profiling. Totally, 246 genes were up-regulated and 35 genes were down-regulated when compared re: AtIPT7-OE AtLEC2-GR with re: AtLEC2-GR , LAFL genes, AGL15, BBM, PLT2, REVOLUTA (REV), CLAVATA 1 (CLV1), WUSCHEL RELATED HOMEOBOX 1 (WOX1), WOX3 and WOX4 were activated  were down-regulated , 40 genes were up-regulated and only one gene was down-regulated , rather than tZ or other cytokinins. Because no callus was obtained in AtIPT3-OE AtLEC2-GR, we are unable to confirm if AtIPT3 has the same effect on the shoot regeneration  and delay plant senescence (data not shown).In this study, multiple endogenous cytokinins could be affected by overexpressing the pendence . The exphenotype , 32. Herncreased . The pheAtLEC2 in tobacco activated many genes encoding embryo proteins and embryo storage proteins, consistent with its regulation roles during embryo maturation [FAD2 and SUS2 suggested that the ectopic LEC2 activity might increase the fatty acid and sucrose accumulation. Overexpression of AtIPTs in the AtLEC2 background differently affected embryogenic callus formation , auxin polar transport genes (PIN1 and PIN2) and auxin signaling genes  in the AtIPT3-OE AtLEC2-GR seedlings implicated that the microenvironment on SAM for callus formation was affected  in AtIPT7-OE AtLEC2-GR and re: AtIPT7-OE AtLEC2-GR were activated when compared with AtLEC2-GR and re: AtLEC2-GR , AtIPT3 (AT3G63110), AtIPT7 (AT3G23630), and AtIPT9 (AT5G20040).The accession numbers of the genes used in this article are as follows: Nicotiana tabacum cv. SR1) were stored in our laboratory. Seeds of Arabidopsis thaliana ecotype Columbia-0 (Col-0) were obtained from the Arabidopsis Biological Resource Center . Seeds were surface-sterilized in 75% ethanol for 30 s and then in 10% (v/v) H2O2 for 10 min, and washed four times in sterile distilled water. Arabidopsis seeds were sown on solid 1/2 Murashige and Skoog (MS) medium containing 1% sugar. After 2 days at 4 °C in darkness, seeds were.Seeds of tobacco  and transformed into Pro35S:AtLEC2-GR transgenic tobacco (Nicotiana tabacum cv. SR1) using leaf discs method [AtIPT3-OE AtLEC2-GR, AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR transgenic tobacco were selected on MS medium containing 50 mg/L kanamycin. Genomic DNA was isolated from the T2 transgenic seedlings. The transgenic lines were further confirmed by PCR amplification using specific primers of AtLEC2, AtIPT3, AtIPT7 and AtIPT9  of s method . The AtIAtLEC2-GR, AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR was transferred to MS medium without DEX and exogenous hormone for shoot regeneration.DEX (25 mM) stock solution was dissolved in absolute ethanol and added to MS medium at a final concentration of 20 μM. Homozygotic transgenic seeds were germinated on MS medium containing DEX for 20 days to obtain embryogenic callus and used for imaging or in vitro shoot regeneration. Callus from Images were captured with OLYMPUS SZX16 microscope and Canon EOS 500D camera. The lengths of hypocotyl and primary root were measured by the measuring tool in OLYMPUS SZX16 microscope. Adobe Illustrator and Photoshop were used for final image arrangement and annotations.AtIPTs influenced embryogenic callus formation, total RNA was isolated from wild type, AtLEC2-GR, AtIPT3-OE AtLEC2-GR, AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR seedlings grown for 20 d on MS medium containing 20 μM DEX for the RNA-sequencing (RNA-seq) experiment. To illustrate the mechanisms that AtIPT7 and AtIPT9 promote shoot regeneration efficiency, we isolated total RNA from AtLEC2-GR, AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR callus after grown on hormone-free MS medium for the RNA-seq experiment. RNA-seq was performed by BGISEQ-500 platform . The data were provided by The Beijing Genomics Institute (BGI). The significantly regulated genes were screened according to Log2 of fold-change and -log10 of the padj  (|Log2 (fold-change) | > 1, padj < 0.1). Identification of significantly  enriched GO categories was done using a web-based tool and database for GO analysis (http://www.geneontology.org/).To gain insight into the mechanism by which Additional file 1:Figure S1. Phenotype of seedlings grown on hormone free MS medium without DEX. Figure S2. Phenotype of mature plants grown in soil. Figure S3. PCR amplified the AtLEC2, AtIPT3, AtIPT7 and AtIPT9 fragments. Figure S4. Seedlings grown on 50 μM DEX condition. Figure S5. Seedlings grown on 20 μM DEX containing medium. Figure S6. Shoot regeneration from the callus. Table S1. Primers used in the study. (DOCX 1921 kb)


Methods: The Cancer Genomic Atlas (TCGA) database was used to determine the prognostic role of mesothelin in PDAC. 99mTc-A1 was evaluated both in vitro in PDAC cells (SW1990 and AsPC-1) and in vivo in an experimental model of mesothelin-expressing PDAC (AsPC-1) in mice. Results: TCGA analysis showed that PDAC patients with high mesothelin expression had a shorter overall survival (P = 0.00066). The binding of 99mTc-A1 was 2.1-fold greater in high-mesothelin-expressing AsPC-1 cells when compared to moderate-mesothelin-expressing SW1990 cells (p < 0.05). In vivo, the 99mTc-A1 uptake was 3.5-fold higher in AsPC-1-derived tumors as compared to a technetium-labeled irrelevant antibody (99mTc-Ctl) (p < 0.01). Conclusions: 99mTc-A1 accurately allows imaging of mesothelin-expressing experimental PDAC tumors. Our experiments paved the way for the development of a companion test for mesothelin-targeted therapies.Mesothelin is a membrane-associated protein overexpressed in pancreatic ductal adenocarcinoma (PDAC). Some mesothelin-targeted therapies are in clinical development but the identification of patients eligible for such therapies is still challenging. The objective of this study was to perform the imaging of mesothelin in mice models of PDAC with a technetium-labeled anti-mesothelin single-domain antibody ( Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, representing the fourth leading cause of cancer-related deaths worldwide in 2018 . MoreoveThe mesothelin gene encodes a 71 kDa precursor protein. It is processed in a shed form known as Megakaryocyte Potentiating Factor, and a 40 kDa GPI-anchored membrane form, which is the mesothelin protein itself . Mesothe99mTc-A1 as a single-domain-based imaging agent used for the phenotypic imaging of membrane mesothelin-expressing breast cancer [99mTc-A1 in imaging mesothelin-expressing PDAC tumors.We recently validated MSLN) when compared to peritumoral  pancreatic tissues (p < 0.05). PDAC patients with high MSLN tumoral gene expression had a significant decreased overall survival when compared to patients with low expression (Pr(>F) = 0.00881)  and their overexpressions were associated with a shorter overall survival (p < 0.01).Tumoral PDAC-derived specimens demonstrated significant increased levels of mesothelin ( tissues A (n = 17pression B (n = 170.00881) C. To fur99mTc-A1 binding was then assessed on these cell lines (99mTc-A1 binding was 2.1-fold higher in AsPC-1 as compared to SW1990 cells (p < 0.05).Through an unbiased in silico approach, mesothelin expression was assessed in 20 PDAC cell lines. An increased, moderated, and reduced mRNA expression of mesothelin was evidenced in AsPC-1, SW1990, and MIAPaCa-2, respectively A. Based ll lines C. 99mTc-99mTc-A1 uptake in mesothelin-positive AsPC-1 cells was readily identifiable, whereas a weak signal was detected using the irrelevant control sdAb  (99mTc-A1 condition displayed a significant greater uptake (P < 0.01)   images are shown in rol sdAb A. This o < 0.01) B. This r < 0.01) C. Linear< 0.001) D. Thus, Despite intense clinical efforts, PDAC remains one of the most aggressive malignancies with a five-year survival rate around 5–7% . The advUsing TCGA datasets, we observed an overexpression of mesothelin only in PDAC-derived specimens in comparison to nontumoral samples. In agreement with other previous studies, high expression of mesothelin predicted shorter overall survival of PDAC patients ,14. The 99mTc-A1, which is a single-domain antibody-derived imaging agent, as an efficient probe in accurately targeting mesothelin-expressing triple-negative breast cancer [99mTc-A1 as an imaging probe of mesothelin-expressing PDAC. Our in vivo experiments showed that 99mTc-A1 enabled the noninvasive visualization of AsPC-1-derived tumors by SPECT imaging at early time points. These results reinforce our study on triple-negative breast cancer, despite the lower 99mTc-A1 uptake of PDAC mesothelin-positive tumors [99mTc-A1 imaging represents a relevant option to visualize other aggressive cancers overexpressing mesothelin such as ovarian and lung cancers, for which the imaging modalities are mainly based on the use of conventional antibodies. However, further in vivo competition experiments using unlabeled A1 would confirm the specificity of 99mTc-A1 uptake by PDAC-derived tumors as previously established in breast cancer [99mTc-A1 and its ability to phenotype mesothelin-expressing tumors in an orthotopic model of PDAC. These results support further preclinical development of 99mTc-A1 and translation to human applications for cancers that overexpress mesothelin.Nuclear imaging represents a highly sensitive and noninvasive imaging modality that could address this challenge. Indeed, we previously validated t cancer ,19,20. T99mTc-A1 for the identification of mesothelin-expressing PDAC that would allow selection of patients who might benefit from mesothelin-targeted therapies that are currently undergoing clinical trials [Future directions of this work would therefore include clinical translation of l trials .mesothelin (MSLN), MKI67, CCNB1, and PCNA were analyzed and compared in tumor (T) and normal tissue (NT) from PDAC-derived specimens using PAAD (Pancreatic adenocarcinoma), TCGA (The Cancer Genome Atlas), and GTEx datasets (T: n = 179 and NT: n = 171) through the available interface Gene Expression Profiling Interactive Analysis (GEPIA) [MSLN, MKI67, CCNB1, and PCNA genes, the KM method was used to estimate survival curves. Hazard ratio (HR) and overall survival were calculated at the best auto-selected cut-off. p-values were calculated using the log-rank test to compare survival curves of high and low gene expression groups and a p-value below 0.05 was considered to be statistically significant.Gene expression levels of  (GEPIA) . The res (GEPIA) . Surviva (GEPIA) ,23,24. TPreprocessed microarray gene expression data deposited in ArrayExpress  were downloaded from the Genomics of Drug Sensitivity in Cancer (GDSC) web page . The gen2 and cultured in DMEM medium  supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.AsPC-1, SW1990, and MIAPaCa-2 cell lines were maintained at 37 °C and 5% COTotal proteins from AsPC-1, SW1990, and MIAPaCa-2 cells were extracted using RIPA buffer . After migration in 12% SDS-polyacrylamide gel, proteins were transferred to nitrocellulose membranes . They were saturated in PBS-Tween 0.1% containing 2% milk at room temperature for 1 h and then probed overnight with the antimesothelin antibody . Membranes were then stripped during 10 min  for β-actin detection .99mTc) using the tricarbonyl method as previously described [99mTc-A1 for 1 h at 4 °C. After five washes in cold PBS, the radioactivity was determined using a γ-counter . Unspecific binding of 99mTc-A1 was determined on MIAPaCa-2 cells and was subtracted from 99mTc-A1 binding on SW1990 and AsPC-1 cells. Results were expressed in counts per minute (CPM).A1 and irrelevant control single-domain antibody (sdAb) were radiolabeled with technetium-99m (3. AsPC-1-tumor-bearing mice were either injected with irrelevant control 99mTc-Ctl (n = 5) or 99mTc-A1 (n = 6). SPECT-CT acquisitions were performed one hour after injection of 42.1 ± 9.0 MBq of 99mTc-sdAbs. 99mTc-Ctl and 99mTc-A1 tumor uptake was expressed in % ID/cm3. Two hours after injection and immediately following SPECT-CT image acquisitions, anesthetized mice were euthanized using CO2 and tumors were harvested and weighed, and tracer activity was determined with a γ-counter . Results were corrected for decay, injected dose, and tumor weight, and expressed as % ID/g.All animal procedures conformed to French government guidelines . They were performed in an approved facility (C385161 0005) under permit APAFIS#3690-2016011916045217 v4 from the French Ministry of Research. Four million AsPC-1 cells were subcutaneously injected into the left flank of five-week-old female Swiss Nude immunodeficient mice (n = 11), in a 2/1 (v/v) PBS/Matrigel (Corning) mix. Tumors were allowed to grow until they reached 200 mmp value below 0.05 was considered significant .Results are expressed as means ± standard deviation and analyzed with GraphPad Prism software . Differences between groups were analyzed using an unpaired Mann–Whitney test for intergroup analysis. Significance of linear correlations was assessed using a Pearson’s test. A 99mTc-A1 allows imaging of mesothelin-expressing PDAC. Our study represents the first step to using this technology as a companion test to select patients eligible for mesothelin-targeted therapies.


MALAT1) in non-small cell lungcancer (NSCLC) and uncover the underlying mechanisms. Accumulating evidences indicate that long non-coding RNAs (lncRNAs) play key roles in cancer. This studyaims to clarify role of the metastasis-associated lung adenocarcinoma transcript 1  assay. Cell transfection was conductedusing Lipofectamine 3000. Cell proliferation was determined with CCK-8 assay. MMP2 and MMP9 expressions weremeasured with Western blot. Cell invasive ability was evaluated by Transwell assay. Starbase 2.0 tool was used topredict targets of MALAT1. Dual luciferase reporter assay, RNA-binding protein immunoprecipitation assay and RNApull-down assay were conducted to confirm the potential direct interaction between MALAT1 and miR-202. In this experimental study, MALAT1 was overexpressed in NSCLC samples and cell lines. High expression of MALAT1 was relatedto large tumor size (>3 cm), poor histological grade, advanced cancer and tumor metastasis in NSCLC. In vitroassays exhibited that knockdown of MALAT1 remarkably decreased A549 cell growth and invasion capacity, whileoverexpression of MALAT1 significantly enhanced NCI-H292 cell proliferation and invasion ability. Next, we verified thatMALAT1 could act as a competing endogenous RNA (ceRNA) by sponging miR-202 in NSCLC and there is a negativecorrelation between MALAT1 and miR-202. Besides, overexpression of miR-202 inhibited cell proliferation and invasiveability in MALAT1-overexpressed cells.MALAT1 gets involved in NSCLC progression by targeting miR-202, indicating that MALAT1 may serve as a novel therapeutic target for NSCLC treatment.This study demonstrated that lncRNA- Lung cancer is the first cause of cancer deathsworldwide, leading to about 1.6 million patients die peryear . Thus, iMEG3 was downregulated in gastric carcinomaspecimens and overexpression of it could repress gastriccancer cell growth and mobility via elevating p53expression. lncRNA CPS1-IT1 was reported to serve astumor suppressor in colorectal cancer and low CPS1-IT1expression indicated poor prognosis (Long noncoding RNAs (lncRNAs) are an emergingclass of transcripts, which is longer than 200 nucleotides(nt). Although lncRNAs are coded by the genome, theyare hardly translated into proteins. Previous researchesrevealed that lncRNAs serve as new regulators, controllinggene expressions epigenetically and post-transcriptionally.They also play crucial roles in modulating chromatindynamics, cell growth, differentiation and development. Increasrognosis .MALAT1), also called as nuclear-enrichedabundant transcript 2 (NEAT2), HCN, LINC00047,NCRN00047 and PRO2853, is an extensively expressedlncRNA, with the length of around 8000 nt  through modulating PI3K/AKT signaling pathway,moderate or poor differentiation, advanced tumor stageand metastasis. Biologically functional experimentsdemonstrated that MALAT1 promoted NSCLC cellproliferation and invasion. Further molecular mechanismsrevealed that MALAT1 could sponge miR-202 withinNSCLC progression.In this study, we observed that lncRNA-Forthy NSCLC tissues as well as correspondingadjacent normal tissues specimens were collected fromGuangzhou Panyu Hospital of Chinese Medicine betweenJune 2015 and July 2018. Patients involved in this studyhad not received any preoperative radiotherapy orchemotherapy. All specimens were identified as NSCLCtissues or normal lung tissues via histopathologicalobservation. After resection, all tissues were dipped inliquid nitrogen promptly and then were stored at -80˚Cfor further studies. All enrolled patients were informedto sign the written informed consent and this study wasapproved by the Ethics Committees of Guangzhou PanyuHospital of Chinese Medicine (license number of ethicsstatement: 2015HW126).2 at 37˚C.In this experimental study, normal lung cell BEAS-2B, NSCLC cell lines  and HEK293T cell wereobtained from ATCC. BEAS-2B cell was culturedin BEBM medium  containing 10% fetal bovine serum . NSCLC cell lines andHEK293T cell were cultured in RPMI-1640 medium supplemented with10% (v/v) FBS. All cells were maintained in a humidifiedatmosphere with 5% COMALAT1was analyzed on a CFX96 real-time thermocycler  by using SsoAdvanced™ Universal SYBR® GreenSupermix . Detection of miR-202 wasperformed using TaqMan microRNA Assay kit  on the CFX96 real-time thermocycler. GAPDH and U6 were considered asendogenous control of lncRNA-MALAT1 and miR-202respectively. Relative expression levels were calculatedby using 2-ΔΔCT method. All primers used in this study arelisted below:Total RNA was extracted from tissue specimens and celllines by using TRIzol reagent  accordingto manufacturer’s protocol and treated with DNase I to remove genomicDNA. cDNA was synthesized with the TranscriptorFirst Strand cDNA Synthesis Kit .For miRNAs, reverse transcription was conducted withTaqMan Micro-RNA Reverse Transcription Kit . Expression level of lncRNA-MALAT1-F: 5′-AGTACAGCACAGTGCAGCTT-3′ R: 5′-CCCACCAATCCCAACCGTAA-3′GAPDH-F: 5′-GGAGCGAGATCCCTCCAAAAT-3′R: 5′-GGCTGTTGTCATACTTCTCATGG-3′miR-202-F: 5′-CCTCCCAGGCTCACGAGGCT-3′R: 5′-GGTGCAGGTGCACTGGTGCA-3′U6-F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′.MALAT1 were quoted from Zuo etal. .Transfection was conducted with Lipofectamine 3000Reagent  in accordancewith the manufacturer’s instruction. The sequence ofsiRNAs against MALAT1 were as follows:siRNAs oligo targeting si-MALAT1: 5′-GAGCAAAGGAAGUGGCUUA-3′si-NC: 5′-CGUACGCGGAAUA CUUCGAdTdT-3′.3 cells/well wereseeded in 96-well plates and cultured overnight. Thecell viability was measured with CCK-8  at different time of culture  following the manufacturer’s instruction.At 24 hours post-transfection, 1×10Total protein was extracted from cell pellet usingRIPA lysis buffer supplemented with protease inhibitors and phosphataseinhibitors (Roche) according to the manufacturer’sprotocol. Concentration of total protein was determinedby using BCA™ Protein Assay Kit . Then, 40 μg of protein per lane wasseparated by 8% sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and transferred topolyvinylidene fluoride (PVDF) membranes. 5% skimmilk was used to block PVDF membranes for 1 hour atroom temperature. Next, membranes were incubatedwith primary antibodies overnight at 4˚C, followed byincubation of secondary antibodies for 1 hour at roomtemperature. Next, protein bands were visualized using theenhanced chemiluminescence system . Primary antibodies, includingMMP2 (1:1000), MMP9 (1:1000) and β-actin (1:1000)and HRP-conjugated secondary antibodies (1:5000) wereobtained from Cell Signaling Technology . β-actin was regarded as the internal control.4 suspended cells in100 μl serum free medium were seeded in upper chamberssmeared on Matrigel . Bottomchambers were filled with 600 μl medium containing 10%FBS. After 48 hours culture, upper chambers were fixed with4% formaldehyde and stained with 0.05% crystal violet.Then, a cotton swab was used to rub away cells on the abovemembrane. The invaded cells through membrane werecounted using optical microscopy.24-well Transwell chambers were purchased from Corning(USA). After 24 hours transfection, 1×10MALAT1 with miR-202binding sites was cloned into the downstream of fireflyluciferase gene in pGL3  to constructpGL3-MALAT1 wild type (WT) and mutant (Mut).HEK293T cells were co-transfected with WT-MALAT1,Mut-MALAT1 reporter gene plasmid or pRL-TK plasmidsand miR-202 mimics or miR-NC with Lipofectamine 3000. The pRL-TK Vectorwas intended for use as an internal control reporter vectorand may be used in combination with any experimentalreporter vector to co-transfect mammalian cells. The pRLTK Vector contains the herpes simplex virus thymidinekinase (HSV-TK) promoter to provide low to moderatelevels of Renilla luciferase expression in co-transfectedmammalian cells. 48 hours later, luciferase activity wasdetermined with a dual-luciferase reporter assay system.Firstly, the full-length 3′-UTR of A Magna RIP RNA binding protein immunoprecipitationkit was obtained from Millipore and the Ago2 antibody was purchased from Abcam. RIP assay was conducted using themagna RIP RNA binding protein immunoprecipitation kitand Ago2 antibody in accordance with the instruction ofmanufacturer. qRT-PCR was used to determine expressionlevel of co-precipitated RNAs.miR-202were synthesized by GenePharma company . 48 hours after transfection with biotin-labeledmiR-NC or biotin-labeled miR-202, the cells werecollected to conduct an RNA pull-down experiment usingPierceTM Magnetic RNA Protein Pull-down Kit  following the manufacturer’sinstruction. lncRNA-MALAT1 level was determinedusing qRT-PCR from the pull-down samples.Biotin-labeled miR-NC and biotin-labeled Statistical analyses were processed with GraphPad Prism6.0 software  and all data wereexpressed as mean ± standard deviation (SD). Student t testor one-way ANOVA was used to determine the differencesbetween two groups or among multiple groups respectively.P<0.05 was considered as statistically significant.MALAT1 indevelopment of NSCLC carcinogenesis, we analyzedlncRNA-MALAT1 expression in 40 paired NSCLC tissuesand pericarcinomatous normal tissues with qRT-PCR. Asshown in Figure 1A, the expression level oflncRNA-MALAT1 was notably higher in NSCLCtissue samples than that in pericarcinomatous normal tissue (P<0.05).To further analyze the relationship betweenlncRNA-MALAT1 expression and clinicalpathological parameters,40 NSCLC patient samples were classified into twogroups in accordance with the median relative quantityof lncRNA-MALAT1. The lncRNA-MALAT1 expressionlevels above the median expression were defined as highexpression while low expression of lncRNA-MALAT1was termed as the expression was below the median level.Results showed that high lncRNA-MALAT1 expressionsignificantly associate with tumor size (>3 cm), moderateor poor differentiation carcinoma, advanced tumor stage and tumor metastases  compared to the normal lung cell BEAS-2B,indicating that lncRNA-MALAT1 may play a promotorrole in NSCLC. Furthermore, A549 cell expressed thehighest level of lncRNA-MALAT1 and NCI-H292 cellexpressed the lowest level of lncRNA-MALAT1, comparedto the other cell lines. Hence, A549 cell was chosenfor silencing lncRNA-MALAT1 and overexpression oflncRNA-MALAT1 was performed on NCI-H292 cell.To investigate the role of lncRNA-tastases , P<0.05.MALAT1expression in A549 cells by transfecting si-MALAT1-1, we silenced lncRNA-MALAT1-1, P<0.05.feration , P<0.05.pression ,P<0.05.ability , P<0.05.MALAT1 in NSCLC, we established NCI-H292cell with overexpression of lncRNA-MALAT1 . Besides, Transwell assay revealed that therelative invasion capacity of NCI-H292 cells in MALAT1-overexpressed group was notably enhanced compared topcDNA3.1 group , to predicttargets of lncRNA-MALAT1. We found that lncRNAMALAT1 has a potential binding site to miR-202 (miR-202 binds to the 3´-UTR of lncRNA-MALAT1 directly. Luciferase activity was markedlyattenuated in HEK293 T cells co-transfected withMALAT1-WT plasmids and miR-202 mimics (P<0.05),while there was no change in the cells co-transfected withMALAT1-Mut plasmids and miR-202 mimics  served as an oncogene in NSCLC. Highexpression of lncRNA-UCA1 predicted short survivaltime and multivariate analysis indicated that UCA1 was anindependent risk parameter of prognosis. Chen et al. (SNHG20) was notably correlated with advancedtumor stage, lymph node metastases and larger tumorsize, as well as poorer overall survival chance. Wang etal.  has been investigatedin NSCLC. Results demonstrated that lncRNA-HITfacilitated NSCLC cell growth through interacting withE2F1 to regulate its target genes (TUG1 (taurineupregulated gene 1) functions as a tumor suppressor inNSCLC compared to corresponding adjacent normal tissues andnormal lung cell BEAS-2B, respectively. This finding wasin accordance with previous studies. Further correlationanalysis demonstrated that high MALAT1 expression waspositively related to large tumor size, poor histologicalgrade, terminal stage of cancer and tumor metastasis.Knockdown of MALAT1 inhibited A549 cell growthand invasion, as well as the expression of MMP2 andMMP9. In contrary, overexpression of MALAT1 elevatedNCI-H292 cell proliferation, invasion ability as well asthe expression level of MMP2 and MMP9. These dataindicated that MALAT1 functions as oncogene in NSCLC,which is in line with previous studies.patients . Accumulin NSCLC . Thestuin NSCLC  showed tCASC2 could function as a ceRNA throughsponging miR-18a in colorectal cancer. In the presentstudy, targets of MALAT1 were predicted by Starbase2.0 (http://starbase.sysu.edu.cn). Then we used dualluciferase reporter gene assay, RIP assay as well as RNApull-down assay to confirm that miR-202 was a directtarget of MALAT1. MiR-202, a new tumor suppressor,is down-regulated in gastric cancer  with mRNAs can actas miRNA decoys. It has been reported that lncRNAs canact as ceRNA by sponging miRNAs in cancer progression. The undc cancer . In addic cancer . miR-202tionally . In prostionally . Sun et tionally  found tho et al.  revealedMALAT1contributes to cancer progression appears to be diversein different cancers. In gastric cancer, MALAT1 wasfound to increase cell viability via modulating SF2/ASF. In ovar pathway. In the MALAT1 could facilitate cellgrowth and invasion via sponging miR-202 in NSCLC.Thus, our research demonstrated a new axis of MALAT1/miR-202, suggesting a feasible therapeutic means forNSCLC treatment.This study elucidated that


We found that HCT116 p53+/+ cells were significantly more sensitive to TRAIL-R-triggering than p53-/- cells. Similarly, A549 lung cancer cells expressing wild-type p53 were more sensitive to TRAIL-R-mediated cell death than their derivatives with knockdown of p53. Our data demonstrate that the contribution of p53 in regulating TRAIL-R-induced apoptosis does not correlate to the levels of TRAIL-Rs at the plasma membrane, but rather to p53-mediated upregulation of Bax, favouring the mitochondrial amplification loop. Consistently, stronger caspase-9 and caspase-3 activation as well as PARP-cleavage was observed following TRAIL-R-triggering in HCT116 p53+/+ compared to HCT116 p53-/- cells. Interestingly, HCT116 p53+/+ cells showed also a more potent activation of non-canonical TRAIL-R-induced signal transduction pathways like JNK, p38 and ERK1/ERK2 than p53-/- cells. Likewise, these cells induced IL-8 expression in response to TRAIL, Mapatumumab or Lexatumumab significantly stronger than p53-/- cells. We obtained similar results in A549 cells with or without p53-knockdown and in the two isogenic colon cancer cell lines RKO p53+/+ and p53-/-. In both cellular systems, we could clearly demonstrate the potentiating effects of p53 on TRAIL-R-mediated IL-8 induction. In conclusion, we found that wild-type p53 increases TRAIL-R-mediated apoptosis but simultaneously augments non-apoptotic signaling.Due to their ability to preferentially induce cell death in tumor cells, while sparing healthy cells, TNF-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL-R1 or anti-TRAIL-R2-specific antibodies are under clinical investigations for cancer-treatment. However, TRAIL-Rs may also induce signaling pathways, which result in malignant progression. TRAIL receptors are transcriptionally upregulated via wild-type p53 following radio- or chemotherapy. Nevertheless, the impact of p53 status on the expression and signaling of TRAIL-Rs is not fully understood. Therefore, we analyzed side by side apoptotic and non-apoptotic signaling induced by TRAIL or the agonistic TRAIL-R-specific antibodies Mapatumumab (anti-TRAIL-R1) and Lexatumumab (anti-TRAIL-R2) in the two isogenic colon carcinoma cell lines HCT116 p53 TRAIL (TNF-related apoptosis-inducing ligand) binds to four plasma membrane-bound receptors TRAIL-R1-4). Two of them, TRAIL-R1 and TRAIL-R2, are capable of inducing apoptosis via their intracellular death domain (DD) and are therefore called death receptors. The two other receptors, TRAIL-R3 and TRAIL-R4, lack a functional DD thus are not able to induce cell death. TRAIL-R3, anchored in the plasma membrane via glycosylphosphatidylinositol, contains neither a transmembrane nor a cytoplasmic domain. Thus, this receptor cannot transmit TRAIL-induced signaling. The cytoplasmic domain of TRAIL-R4 is able to induce several non-apoptotic signal transduction pathways but possesses a truncated, non-functional DD. Both TRAIL-R3 and TRAIL-R4 were proposed to negatively regulate TRAIL-induced apoptosis via direct interaction and/or ligand competition with the pro-apoptotic receptors TRAIL-R1/R2 [-4. Two oUpon TRAIL ligation, TRAIL death receptors assemble at their intracellular DD the death-inducing-signaling-complex (DISC) composed of FAS-associated protein with death domain (FADD) and pro-caspase-8/10 . ProximiκB, MAP kinases, AKT, PKC and Src, which may lead, particularly in apoptosis-resistant cells, to increased proliferation, invasion and metastasis [TRAIL´s ability to preferentially kill tumor cells, while sparing healthy cells, makes it a promising weapon for targeted tumor therapy ,7. Howevtastasis –13.TRAIL receptors are known transcriptional targets of the tumor suppressor protein p53, and chemotherapeutic agents potentiate cell death in wild-type p53-expressing tumor cells by enhancing TRAIL-R1/R2 expression at the plasma membrane ,15. TherIn the majority of human cancers, p53 is either mutated or inactivated via complexing with inhibitory proteins like Mdm-2. For such tumors, induction of TRAIL-R-mediated cell death, as a p53-independent pathway, may represent a promising alternative therapeutic strategy. There are several available agonists of TRAIL-Rs, including recombinant human TRAIL or agonistic TRAIL-R-specific antibodies . HoweverIn the present study, we investigated side by side the impact of wild-type p53 on apoptotic and non-apoptotic TRAIL-R signaling following treatment with TRAIL or agonistic, receptor-specific antibodies.+/+ (WT) and HCT116 p53-/- (KO) were kindly provided by Bert Vogelstein and described previously [+/+ and RKO p53-/- were purchased from Horizon Discovery, Cambridge, UK. HCT116, RKO, and lung cancer A549 cells all were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine and 1 mM sodium pyruvate. To the cell culture medium for HCT116 p53-/- cells, additionally Geneticin (400 μg/mL) and Hygromycin B (100 μg/mL) were added. The antibiotics were omitted when the cells were seeded for subsequent stimulations. Cells were stimulated with recombinant human TRAIL  or with the agonistic anti-TRAIL-R1- or anti-TRAIL-R2 antibodies Mapatumumab and Lexatumumab , respectively. To inhibit caspases, zVAD-fmk  was added to the cell culture medium 1 h prior to the treatment with TRAIL-R-targeting agents. To knock down the expression of p53 in A549 cells, cells were transfected with ON-TARGET plus TP53 or control siRNA  using a LipofectamineTM RNAiMAX transfection reagent  for 72 h.The human colon cancer cell lines HCT116 p53eviously . The col+/+ / p53-/-) and RKO (p53+/+ / p53-/-) cells were seeded (2x104/ well) in 96-well plates grown for 24 h and stimulated with TRAIL, agonistic anti-TRAIL-R1- or anti-TRAIL-R2 antibodies and zVAD-fmk for 24 h. Cell viability was assayed by crystal violet staining as described previously [HCT116 (p53eviously .+/+ / p53-/-) cells were seeded in 6-Well plates (2x105/ well) grown for 24 h and stimulated with TRAIL or agonistic anti-TRAIL-R1- or anti-TRAIL-R2 antibodies for 24h. Cell death was evaluated by staining of cells with 2 μg/mL of propidium iodide (PI) followed by determination of membrane integrity via flow cytometry by FACSCalibur . A population size of 30.000 cells was regarded as representative for data evaluation using FlowJo v10 .HCT116 (p53+/+ / p53-/-) and A549 (Ctrl-si / p53-si) cells were seeded in 6-Well plates (2x105/ well) grown for 24 h and stimulated with TRAIL or agonistic anti-TRAIL-R1- or anti-TRAIL-R2 antibodies for 12 h (HCT116 p53+/+ / p53-/-) or 24 h (A549 Ctrl-si / p53-si). DNA fragmentation was measured using propidium iodide staining followed by flow cytometry analysis as described previously [HCT116 , anti-human TRAIL-R2 , anti-human TRAIL-R3  or anti-human TRAIL-R4 , all purchased from R&D Systems GmbH, Wiesbaden, Germany. Respective isotype control stainings were performed with APC-conjugated mouse IgG1 (clone #11711) and mouse IgG2B (clone #13303) antibodies (both from R&D Systems GmbH). Finally, cells were washed twice in cold MACS buffer, resuspended in cold MACS buffer supplemented with 1% PFA and measured within 24 h using a FACSCalibur . A population size of 30,000 cells was regarded as representative for data evaluation using FlowJo v10 .Cell surface expression levels of TRAIL receptors were analyzed by flow cytometry. Briefly, cells were detached from culture dishes by treatment with Accutase . Afterwards, cells were washed with cold MACS buffer  and FcR-blocking was performed with human FcR blocking reagent  according to the manufacturer’s instructions. For single stainings of TRAIL receptors, 2 x 106 cells were seeded on 150 mm plates and cultured for 36 h. After removal of the medium, cells were washed twice with ice-cold PBS and incubated with 10 ml Sulfo-NHS-SS-Biotin (0.24 mg/ml in PBS) while being gently shaken on ice for 15 min. Then, the cells were washed with ice cold PBS once and incubated for 10 min with 10 ml quenching solution . Thereafter, the cells were washed with ice cold PBS and lysed with RIPA buffer supplemented with Complete Protease Inhibitor Cocktail and PhosphoStop . Biotinylated proteins were isolated from 400 μg of whole cell lysates using streptavidin conjugated magnetic beads  overnight on a roller at 4°C. The beads were washed three times with TBST, resuspended in 25 μl 2 x Laemmli buffer and boiled for 7 minutes. Finally, buffer containing precipitated proteins was magnetically separated from the beads and 5 μl were loaded onto SDS gels.4 x 10Cells were lysed in RIPA buffer supplemented with Complete Protease Inhibitor Cocktail and PhosphoStop  and Western blot analyses were performed as described previously . PrimaryCells were homogenized with QIAshredder  and total RNA was isolated with an RNeasy Mini Kit . The expression of IL-8 was studied by RT-PCR using TaqMan assays  and a 7900HT Fast RT-PCR system (Thermo Fisher Scientific). The expression levels were calculated relative to the expression of the housekeeping gene TATA-binding protein (TBP) by ΔΔCT method. Primers were purchased from Thermo Fisher Scientific (TBP (Hs00427620_m1) and IL8 (Hs00174103_m1)).The IL-8 levels were determined in cell culture supernatants via ELISA  according to the manufacturer’s protocol.Statistical analyses were performed using SigmaPlot v12.5 . First, data were tested for normality and equal variance by Shapiro-Wilk and Equal Variance test, respectively. For comparison of two-groups comprising parametric distributed datasets, t-test was applied. Two groups of non-parametric datasets were analyzed with Mann-Whitney Rank Sum test. Statistically significant differences between the groups were assumed at p-values < 0.05 and marked with an asterisk (*).+/+ and p53−/− cells by flow cytometric analyses. As shown in −/− cells compared to p53+/+ cells. In contrast, the expression of TRAIL-R3, albeit much less pronounced, was higher at the cell surface of p53+/+ cells than p53−/− cells. The surface expression levels of TRAIL-R2 and TRAIL-R4 remained unchanged. To validate these data by an independent approach, we biotinylated cell surface proteins and subsequently analyzed the levels of biotinylated TRAIL-Rs in whole cell lysates by Western blotting. The results shown in -/- when compared to HCT116 p53+/+ cells and show that the levels of TRAIL-R3 are slightly downregulated.Since TRAIL receptors are known transcriptional targets of p53, we first asked whether the p53 status might affect their expression in HCT116 cells. For this purpose, we compared the cell surface levels of TRAIL-R1/R2/R3/R4 in HCT116 p53+/+ and p53−/− and in addition in a lung cancer cell line A549 with and without transient knockdown of p53. Flow cytometric analyses of TRAIL-R expression at the plasma membrane revealed that in these cell lines, loss of p53  or TRAIL-R2 (Lexatumumab) and determined cell viability by crystal violet staining  polymerase 1 (PARP-1), more prominent  levels of TRAIL death receptors in untreated cells, we found that while the expression of TRAIL-R1 remained unchanged between the cell lines, p53TRAIL-R1 . In contatumumab .Interestingly, an additional protein band of approximately 45 kDa, could be detected by p53-specific antibodies in treated but not in untreated control cells, suggesting that it might represent a cleavage product of full length p53 .-/- cells compared to HCT116 p53+/+ cells . Of note, HCT116 cells signal preferentially via TRAIL-R1 [Interestingly, the increased level of TRAIL-R1 at the cell surface of HCT116 p53view see ). Thus, view see . It is tTRAIL-R1 . Thus, i+/+ cells than in p53-/- cells and of Bax in A549-Ctrl cells compared to A549-p53-KD cells.The pro-apoptotic role of p53 in TRAIL-mediated cell death might be explained by the fact that several targets of p53 like Bax, APAF1 and Bid are important mediators of TRAIL-induced cell death –34. Acco+/+ cells compared to p53-/- cells. Although analysis of the TRAIL-R2 expression at the plasma membrane showed no differences in both cell lines, intracellular TRAIL-R2 might delocalize from the cytoplasm to the plasma membrane and contribute to stronger apoptosis induction in p53+/+ cells. In addition to localization at the plasma membrane, TRAIL-R1 and TRAIL-R2 are often found intracellularly in many cells types, including HCT116 cells [Moreover, the basal level of TRAIL-R2, which is also a p53 target, was slightly enhanced in p5316 cells ,36.+/+ compared to HCT116 p53-/- cells.Recently, it has been demonstrated that TRAIL and chemotherapeutic drugs may cause the cleavage of p53 into pro-apoptotic fragments via activation of caspase-3. In HCT116 and other p53 wild-type expressing cells, these p53-fragments translocated into the mitochondria leading to the release of cytochrome c and improvement of TRAIL/chemotherapeutics-induced apoptosis . Since w+/+ cells responded to TRAIL receptor-triggering with stronger induction of pro-inflammatory pathways than p53-/- cells. We observed a similar p53-mediated enhancement of pro-inflammatory IL-8 expression in two other cell lines, RKO and A549, suggesting the more general character of these observations. The capability of TRAIL to induce IL-8 expression has been demonstrated by us and others for different tumor cells [In addition to stronger activation of apoptotic signaling, HCT116 p53or cells ,38–40. Ior cells . RecentlOur data showing the function of p53 as a factor potentiating the non-apoptotic, pro-inflammatory signaling of TRAIL death receptors are potentially of clinical relevance since it has been reported that 5-FU treatment leads to the induction of TRAIL expression in NK cells in vivo . It is w+/+ expressing cells involves the risk of promoting pro-survival and metastatic pathways in response to TRAIL or TRAIL-R-agonistic antibodies. Whether TRAIL-R-mediated apoptotic or non-apoptotic signaling dominates and which factors influence the switch between these pathways will be the subject of further investigation.In summary, our findings provide that although wild-type p53-expressing cells are more sensitive towards TRAIL-R-mediated cell death than p53-KO or p53-KD cells, TRAIL-R-mediated pro-inflammatory pathways are concomitantly also stronger induced in these cells. Thus, TRAIL-R-targeted anti-cancer therapy in p53S1 Fig+/+/ p53-/- and RKO p53+/+/ p53-/- cells were biotinylated with Sulfo-NHS-SS-Biotin. Cells were lysed with RIPA buffer and biotinylated proteins purified via streptavidin conjugated magnetic beads from equal μg of protein lysates. TRAIL receptor expression was analyzed by western blot of purified proteins using TRAIL-R-specific antibodies. Actin serves as a loading control for biotin pull-down.Extracellular regions of surface proteins on the cell surface of HCT116 p53(TIF)Click here for additional data file.S2 Fig+/+ and RKO p53-/- cells were stimulated either with TRAIL (200 ng/ml), Mapatumumab (10 μg/ml) or Lexatumumab (10 μg/ml) for 24 h with or without zVAD-fmk (20 μM). Cell viability was determined by crystal violet staining (A). Results are shown ± SD of three biological replicates (n = 3).RKO p53(TIF)Click here for additional data file.S3 Fig+/+ and RKO p53-/- cells were stimulated either with TRAIL (200 ng/ml), Mapatumumab (10 μg/ml) or Lexatumumab (10 μg/ml) for 3 h. Whole cell lysates were analyzed for the phosphorylation/activity status and overall expression of various proteins associated with TRAIL-mediated non-apoptotic signaling pathways by Western blot (A). Blots are shown for one representative experiment out of three performed.RKO p53(TIF)Click here for additional data file.


The transgenic rescue animal displayed defects in gonad migration and tail morphology. Also, the mutant animals produced a high number of males, suggesting that the Y to E mutation in βpat-3 integrin causes a phenotype similar to that of Him mutant. Further analyses revealed that males of pat-3(Y804E) and him-4/hemicentin share additional phenotypes such as abnormal gonad and unsuccessful mating. A pat-3 transgenic rescue mutant with a non-polar phenylalanine (F) in NPXY, pat-3(Y792/804F), suppressed the high male number, defective mating, inviable zygote, and the abnormal gonad of him-4 mutants, indicating that Y to F mutation in both NPXY motifs suppressed the him-4 phenotypes. This finding supports the idea that the ECM determines the activation state in integrin NPXY motifs; him-4/hemicentin may directly or indirectly interact with integrins and maintain the NPXY non-charged. Our findings provide new insight into a suppressive role of an ECM molecule in integrin NPXY phosphorylation.Integrin, an αβ heterodimeric cell surface receptor for the extracellular matrix (ECM), carries two tyrosine phosphorylation motifs in the cytoplasmic tail of the β subunit. NPXY (Asn-Pro-x-Tyr) is a conserved tyrosine phosphorylation motif that binds to the phospho-tyrosine binding (PTB) domain. We generated a tyrosine to glutamic acid (E) mutation to modify tyrosine (Y) into a negatively charged amino NPXY in the β Integrin is a cell surface receptor for the extracellular matrix (ECM), playing a significant role in cell adhesion and tissue organization. Upon binding to the ECM, integrin links ECM components to cell signaling and cytoskeletal molecules via its cytoplasmic tail. Notably, the β integrin cytoplasmic tail contains conserved tyrosine phosphorylation sites. These are NPXY (tyrosine phosphorylation) motifs essential for recruitment of focal adhesion proteins such as talin  and kindIntegrin is an excellent molecule to study the function of the NPXY motif, because two NPXY motifs are conserved in many species and arranged in the cytoplasmic tail of β integrins  Figure . For exahave examined the function of NPXY in the β integrin cytoplasmic tail and found that the Y to F mutation sustains integrin functions while the mutation to alanine (A) abolishes the cellular function of β integrin , suggestCaenorhabditis elegans is an excellent genetic model organism to study integrin function, having two α and one β chains (pat-3β integrin (PAT-3) is expressed in muscles, gonads, and neurons and plays essential roles in tissue organization  phenotypes. This demonstrated that the role of integrin in postembryonic tissues justifies further analyses of β integrin cytoplasmic tail  is replaced by a negatively charged/acidic amino acid, glutamic acid (E). Although the charged E side chain appears different from the phospho-tyrosine, the Y to E mutation has been used as an analogous substitution for the phospho-tyrosine  mutation rescued embryonic lethality of pat-3 null and created viable transgenic animals. However, it also displayed Him  and defective male mating, which is similar to that of him-4 mutant alleles.There have been several reports about the NPXY motif in hrodites . The DEPormation , suggesttyrosine . Reszka  binding . In our him-4, encoding a large ECM protein similar to human hemicentin I, cause defective cytokinesis of mitotic germ cells and consequently lead to chromosomal losses as well as production of abnormal males with a mating defect (pat-3(Y792/804F), suppressed Him, abnormal gonad, and unsuccessful mating of him-4/hemicentin mutants, suggesting that HIM-4/hemicentin interacts with pat-3 integrin and modulates the charged state of the NPXY. This suggests that NPXY motifs in β integrin play a vital role in its function, which impacts many aspects of development and interaction between cells and their surroundings.g defect . Anotherg defect . Our genCaenorhabditis Genetics Center at the University of Minnesota, Minneapolis, MN. Mutant lines used in this study include RW3600 qC1 pat-3(st564)/dpy-19(e1259) glp-1(q339) III, raIs8[unc-112:GFP + rol-5(su1006)] (lon-2(e678) him-4(e1267) X, and him-4(rh319) X. Transgenic pat-3 rescue animals used in this study include mwEx443[pat-3 (+)], mwEx35[pat-3 (Y792F)], mwEx31[pat-3 (Y804F)], mwEx32[pat-3 (Y792/804F)], and kqEx804[pat-3(Y804E)] (this study), which are described in Wild-type Bristol strain, N2, and mutant strains were purchased from the su1006)] , lon-2(eC. elegans strains were cultured on NGM plates seeded with OP50 (mwEx35[pat-3 (Y792F)]; him-4(e1267), mwEx31[pat-3 (Y804F)]; him-4(e1267), and mwEx32[pat-3 (Y792/804F)]; him-4(e1267), were generated by crossing homozygote or heterozygote males from each transgenic line to lon-2(e678) him-4(e1267) ] animals were fed with him-4 RNAi bacteria, a clone F15G9.4 (X-4F24) from the C. elegans RNAi library . The localization of UNC-112:GFP was observed 72 h after feeding.All ith OP50 . Four do4(e1267)  hermaphr4(e1267) . For Himpat-3(Y804E) mutation construct, an overlap extension method to create mutations in the 787NENPIY792 or 799FKNPVY804 sequence was performed using pPAT3-PB12K, wild-type pat-3 gene, as a template. The pPAT3-pPB12K genomic DNA, containing 11799 bases of pat-3 gene, was cloned between PvuII and PstI in pSP73 , described in the previous study (To generate the us study . PrimersGAG GCT GGA AAA GCC -3′PAT3 Y2EFW3: 5′- CCA GTA CTC TAC TGG -3′PAT3 Y2ERV3: 5′- GGC TTT TCC AGC GA GAA ACA GGC CAC G -3′PAT3 EJ Y1EFW1: 5′- CCC AAT CCTC GAT TGG G -3′PAT3 EJ Y1ERV2: 5′- CGT GGC CTG TTT PAT3 FORWARD(FW)3: 5′- TGC TAG CCC TCC ACC TGT CCC TGT G -3′PAT3 REVERSE(RV)5: 5′- CCA CAC GGT AAA TAG G -3′EcoRI and cloned into pSP73 vector for sequencing to confirm mutations. The sequence confirmed DNA construct was digested with MscI and EcoRI, and the fragment was inserted back into pPAT3-PB12K (sur-5:GFP DNA (pat-3(st564)/qC1 dpy-19(e1259) glp-1(q339) III strain  was used for further studies. Alternatively, 50 mg/ml of pPAT3(Y792E) or pPAT3(Y804E) DNA was also microinjected into N2 animals to measure the dominant effect of the transgenes. Green F2 animals were isolated and were studied for analysis.To perform overlap extension, two polymerase chain reactions (PCR), PAT3FW3 - Y2ERV3 (pair 1) and Y2EFW3 – PAT3RV5 (pair 2), were prepared. Two amplified resultant DNA molecules were then mixed for the second round PCR as overlap templates for PAT3FW3 and PAT3RV5 primers. The second round DNA was digested with T3-PB12K ; complet:GFP DNA  in TE buI strain  and seleonstruct . The resCoolSnap ES  and analyzed with Metavue software  or NIS Elements . For DIC or fluorescence live samples, animals were fed with 2% NaN3 or 5 mM levamisole solution on agarose pads and mounted on the Nikon TE2000-U or Eclipse Ni-U microscope. Samples were examined and analyzed for defects using a 20X or 40X Plan Fluor objective lens. Images were obtained using Coolsnap ES or DYNO monochrome camera  and analyzed using Metavue  or NIS Elements  software.To examine the muscle cytoskeleton, animals were collected and placed on poly-L-lysine coated slides and fixed with methanol and acetone for 5 min each at −20°C. Fixed worms were treated with rhodamine-conjugated phalloidin  for 2 to 3 h at room temperature. Washed samples were mounted on the Nikon TE2000-U epifluorescence microscopes. Images were captured using a For DNA staining, animals were washed off the plate and fixed with methanol in −20°C. The next day fixed worms were treated with ethanol series from 95–30%. After the final alcohol treatment, isolated animals were treated with 0.1 μg/ml of DAPI  in M9 buffer for more than 4 h with rotation. After three washes, samples were observed under the microscope.Animals were stained with MH25, anti-PAT-3 antibodies  , and goachi-squared likelihood ratio test  construct was injected into a null allele, pat-3(st564), to generate viable transgenic rescue lines with potential mutant phenotypes (kqEx804[pat-3 (Y804E)], the transgenic rescue animal, showed gonad migration defects. During gonad morphogenesis, two distal tips cells (DTC) at the polar ends of hermaphrodite gonad navigate the formation of the mirror image U-shaped tubular gonad (n = 188) (pat-3(Y804E) displayed abnormal turns and an irregular trajectory of distal arms (pat-3(+) wild-type rescue, pat-3(Y804E) animals displayed abnormal tails with irregular tapering at the end, 81.4% (n = 97)   of gonadtal arms . Adult atal arms . The distal arms . These d(n = 97) , of herm(n = 97) .792 was also mutated to NPIE792; and a pPAT3(Y792E) genomic DNA construct was microinjected into pat-3(st564)/dpy-19(e1259) glp-1(q339) III, a balanced pat-3 null , was also created; it showed defects in tissues such as the hermaphrodite gonad, vulva, and tail caused from dominant effects of the pat-3 mutant transgene  transgenic recue animals displayed an unusually high number of males, 4.3% (n = 1179), resulting in a Him  phenotype  males were examined for mating , we found no signs of mating success  males led us to investigate the cause of the mating defect, which can be caused by failed gonadogenesis (pat-3(Y804E) male gonad was examined under DIC microscopy. Distal gonad migration appeared abnormal; the migration path of the cell failed to show a typical pattern. Instead, the gonad tube appeared to make irregular turns (n = 59) (pat-3(Y804E) male tails showed abnormal rays and spicules (pat-3(+) animals (pat-3(Y804E) males were also stained with DAPI to examine the nuclear morphology of the male gonad. The majority of males showed a typical gonad appearance but some showed ectopic puncta; staining of ectopic sperm nuclei was dispersed in the body cavity.The unsuccessful mating of ogenesis  or abnorogenesis . The patar turns , 4B. The(n = 59) . In addispicules ; most faspicules . It shou animals . Next, ppat-3(+) (pat-3(Y804E) male gonads displayed undifferentiated germ cells located posterior to sperm (pat-3(+) or N2 sperm nuclei normally appear posterior to the undifferentiated germ cell nuclei in nuclear staining (pat-3(Y804E) male gonad, however, displayed unique germ cell positioning, suggesting that the proximal end of the gonad failed its posterior migration or failed to fuse firmly to the rectum.In contrast to germ cells being located anterior to sperm in pat-3(+) , pat-3 transgenic line suggested that pat-3(Y804E) defects could be analogous to the phenotype of him-4 alleles  (him-4(e1267) mutant animals displayed Him, unsuccessful mating, and tail abnormality comparable to that of pat-3(Y804E) , a him-4 null allele  and pat-3(Y804E), however, showed muscle filament appearance comparable to that of pat-3(+) animals  suggested that pat-3 is linked to him-4 in gonad formation and function.The phenotypes of the  alleles . The himcentin I ; him-4 a3(Y804E) . The him3(Y804E) , 2. The al gonad , ectopical gonad , and an al gonad . We alsol allele ; rh319 s animals . The simhim-4 alleles and pat-3(Y804E) suggested that the wild-type him-4 affects tyrosine (Y) at the NPVY804 motif to modulate integrin functions. In order to compare the role of NPXY mutations, we also reexamined the phenotype in non-polar NPXY mutants, pat-3(Y792F), pat-3(Y804F), and pat-3(Y792/804F) transgenic rescue animals from our previous analyses (pat-3(+). Male tail morphology appeared wild-type in pat-3(Y792F) and pat-3(Y792/804F), comparable to that of N2. The pat-3(Y804F) animals, however, showed minor defects in tail morphology (pat-3(Y804E).The phenotypic similarity between analyses . In all rphology . Despiterphology , howeverrphology . In addirphology , althoughim-4 in NPXY modulation, the double mutants, pat-3(Y792F); him-4(e1267), pat-3(Y804F); him-4(e1267), and pat-3(Y792/804F); him-4(e1267) were generated. Our analysis showed that the male percentage in pat-3(Y792F); him-4(e1267) and pat-3(Y804F); him-4(e1267) were comparable to him-4(e1267) (pat-3(Y792/804F); him-4(e1267) was much lower than him-4(e1267). Similar analysis was performed with the him-4(rh319) null mutant; the male percentage of pat-3(Y792/804F); him-4(rh319) double showed no signs of male. This suggested that pat-3(Y792/804F) suppresses the Him phenotype of him-4(e1267) and him-4(rh319) to a significantly low level (In order to test the role of 4(e1267) , while tow level .pat-3(Y792F); him-4(e1267) and pat-3(Y804F); him-4(e1267) showed increased abnormal gonad (n = 40) and 81.3% (n = 53), respectively, which was significantly higher level than that of the him-4 alleles ; him-4(e1267) animals showed the defects in 11.6% (n = 69), which was lower than that of the him-4 allele (n = 64). Male tail morphology was also examined (pat-3(Y792F); him-4(e1267) and pat-3(Y804F); him-4(e1267), showed significantly increased in abnormal male tails with deformed genitals such as protruded spicules and irregular fans, 90% (n = 40) and 88.7% (n = 53), respectively ; him-4(e1267) animals appeared as low as 11.6% (n = 69), suggesting that pat-3(Y792/804F) suppressed the tail defects of him-4. We also measured male mating; analyses of the double mutants revealed that pat-3(Y792/804F); him-4(e1267) were able to suppress him-4 male mating phenotype. Only pat-3(Y792/804F); him-4(e1267) males among other double mutants were able to mate with hermaphrodites and produce progeny from mating  due to aneuploidy of autosomal chromosomes (him-4(e1267) and him-4(rh319) animals displayed 30% (n = 1335) and 9.6% (n = 116) egg hatching, respectively. The pat-3(Y792F), pat-3(Y804F), pat-3(Y804E), and pat-3(Y792/804F) animals were also comparable to the hatching rate of him-4(e1267) mutant animals (pat-3(Y792F); him-4(e1267), pat-3(Y804F); him-4(e1267), pat-3(Y792/804F); him-4(e1267), and pat-3(Y792/804F); him-4(rh319). Introduction of him-4 into pat-3 Y to F single mutants exacerbated hatching defects (pat-3(Y792/804F); him-4(e1267) eggs was comparable to that of N2 and pat-3(+). The hatching of pat-3(Y792/804F); him-4(rh319) was significantly increased compared to the rate of him-4(rh319)  , suggest804 motif of βpat-3 integrin cytoplasmic tail was analyzed. The Y to E mutation inducing negatively charged NPXY introduced to the distal motif fails to affect the muscle organization but causes defects in the gonad and tail. This mutant pat-3 integrin also shows a Him phenotype undescribed in previous integrin studies. The phenotypes of pat-3(Y804E) animals resemble defects in him-4 alleles. Double mutant analysis reveals that non-polar pat-3 mutants, pat-3(Y792/804F), suppresses him-4 phenotypes, whereas pat-3(Y792F) or pat-3(Y804F) enhances the gonad phenotype of him-4, suggesting that HIM-4/hemicentin interacts with pat-3 integrin and prevents the chemical modification of both NPXY motifs in the cytoplasmic tail of PAT-3.In this study, the function of the distal NPVYhim-4 mutants, the Him phenotype is a result of aberrant cellularization in mitotic germ cells, which mainly occurs due to incomplete constriction of the rachis (him-4. It appears that the Him phenotype is only linked to pat-3(Y804E), although two Y to F mutants, pat-3(Y804F) and pat-3(Y792/804F), appear to have a higher male number than pat-3(+). Our ANOVA testing and a post hoc multiple comparison indicates that the male percentage of pat-3(+), pat-3(Y792F), pat-3(Y804F), and pat-3(Y792/804F) are not significantly different from each other  mutation (pat-3(Y804E) or him-4 contains ectopic sperm in the vas deferens or in the body cavity, which is undescribed in previous pat-3 integrin studies  phenotype that is similar to the gonad defects of pat-3(Y804E) or him-4 mutant. In these mutants, the gonad linker cell fails to die and be engulfed by U lineage (U.lp and U.rp) epithelial cells to make an opening to the cloaca (pat-3(+) along with other Y to F mutant males  and Y to F mutant males display a low percentage of tail defects. However, these males with a tail defect fertilize hermaphrodites and produce progeny , significantly suppresses Him and mating defects, gonad morphology defects, and embryonic lethal phenotypes of the him-4 allele ; him-4(e1267) and pat-3(Y804F); him-4(e1267) males (pat-3(Y792F); him-4(e1267) may increase negatively charged NPVY804 locally, while there is the similar increase of negatively charged NPIY792 in pat-3(Y804F); him-4(e1267). The him-4(e1267) and him-4(rh319) phenotypes are significantly suppressed in pat-3(Y792/804F); him-4(e1267) and him-4(rh319) double mutants. In this case, both NPXY motifs are kept from the modification, which is similar to default condition, uncharged NPXY motifs.The double mutant analyses indicate that non-polar integrin, 4 allele –3. We wo7) males , 3 are pOur new findings provide valuable information about the function of NPXY motifs in β integrin. They provide us with new insights into how cell-matrix interactions modulate the conserved phosphorylation motifs in the integrin tail. This information will help us to understand integrin and hemicentin functions in many species. Finally, because integrin has been implicated in human pathology such as tumor growth, inflammation, and other diseases, this foundational research on the interaction between β integrin and hemicentin may lead toward gains in research related to human health.The datasets generated for this study are available on request to the corresponding author.ZQ, PS, JA, and E-JY performed the experiments. PS performed the statistical analyses. ZQ and PS wrote experimental procedures and figure legends, and generated figures and 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.


Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogenesis. The mitochondrial derivatives run along the entire flagellum and provide structural rigidity for flagellar movement, but its precise function and organization is incompletely understood. The two mitochondrial derivatives differentiate and by the end of spermatogenesis the minor one reduces its size and the major one accumulates paracrystalline material inside it. The molecular constituents and precise function of the paracrystalline material have not yet been revealed. Here we purified the paracrystalline material from mature sperm and identified by mass spectrometry Sperm-Leucylaminopeptidase (S-Lap) family members as important constituents of it. To study the function of S-Lap proteins we show the characterization of classical mutants and RNAi lines affecting of the S-Lap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the S-Lap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that S-Lap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that S-Lap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm. Drosophila melanogaster sperm and identified Sperm-Leucylaminopeptidase (S-Lap) family members as a major constituent of it. We characterized mutant alleles of S-Lap genes and showed that their male sterile phenotype is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. In this study we provide the first experimental proof that S-Lap proteins are important structural constituents of the paracrystalline material of the Drosophila melanogaster’s sperm. Our results could contribute to the better understanding of the formation of special ordered protein structures, like paracrystalline material, and filamentous enzymes.Mitochondria are essential organelles of developing spermatids, but many of the factors that necessary to mitochondrial differentiation, elongation and structural composition during spermatogenesis need to be identified. In almost all insect species there are two mitochondrial derivatives in mature sperm. One or both mitochondrial derivatives accumulate paracrystalline material; however, its exact molecular composition and function are still unresolved. We purified paracrystalline material from  D. bifurca, 24.29 mm in D. kanekoi or 23.3 mm in D. hydei  = nos-phiC31\int.NLS}X; P{y[+t7.7] = CaryP}attP2 fly lines.Transgenic constructs were injected into w1118 virgin females. Fourteen days after crossing, hatched progeny was counted in every tube.For fertility tests, individual males (minimum 30/genotype) were crossed with three Drosophila melanogaster species restriction (42462 protein sequences), each entry was concatenated with a randomized sequence. The following search parameters were applied: mass accuracy: 5 ppm for precursor ions and 0.6 Da for fragment ions; enzyme: trypsin with maximum one missed cleavage site; fixed modifications: carbamidomethyl; variable modifications: Met oxidation, pyroGlu formation from N-terminal Gln residues and acetylation of protein N-terminus; instrument: ion trap. The following acceptance criteria were applied: score, 22 and 15; E-value, 0.01 and 0.05 for protein and peptide identifications, respectively.For proteomic analysis and immunoblotting 50 seminal vesicles or 20 pairs of testis of 10 days old flies were dissected and homogenized in 100 μl modified RIPA buffer , 1 mM PMSF). Cellular debris was pelleted at 2000 rpm for 2 minutes, and then the supernatant was centrifuged at 15000 rpm for 15 minutes at 4°C, removed and mixed with 20 μl 6xLaemmli sample buffer. The insoluble pellet was washed two times with 0.75 M Tris-HCl (pH 7.6) and centrifuged at 15000 rcf for 15 minutes, then suspended in 120 μl 1xLaemmli buffer. Samples were separated on 10% SDS polyacrylamide gels (Bio-Rad). For proteomic analysis, the gel was stained with Coomassie Brilliant Blue then destained with the mix of 40% ethanol and 10% acetic acid. The 50-65 kDa region of the soluble and insoluble sample were subjected to in-gel digestion as described in Migh et al. . BrieflyFor immunoblotting, the protein samples were fractionated by SDS-PAGE and transferred to PVDF membrane. Blocking and antibody incubations were carried out in Tris-buffered saline (Sigma-Aldrich) with 0.05% Tween-20 (TBST) containing 4% nonfat dry milk. Primary antibodies were anti-beta-tubulin E7 , S-Lap1 antibody (1:1000 dilution) and anti-His antibody . HRP-linked secondary antibodies (Millipore) were used at a dilution of 1:5000. After incubation with the antibodies, blots were washed in TBST and imaged on X-ray film using an ECL detection kit .Drosophila grsm, S-Lap1 and S-Lap6 were inserted into the pDEST17 plasmid in frame with the N-terminal polyhistidine-tag using the Gateway system (Thermo Fisher Scientific). The constructs were transformed into chemically competent E. coli SixPack strain [xg, 4°C, 15 min), lysed by sonication in ice-cold buffer containing 50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.1% Triton X-100 and 5% 2-mercaptoethanol (Buffer-1) and centrifuged  to pellet debris. The clarified lysate was incubated with Ni-NTA agarose beads (Qiagen) for 2h at 4°C. Beads were washed twice in Buffer-1, twice in Buffer-2 (Buffer-1 supplemented with 200 mM NaCl) and twice in Buffer-1 followed by the elution of the recombinant proteins in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl and 300 mM imidazole. Eluates were concentrated and dialyzed against a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MnCl2 and 50 μM ZnCl2. Purified proteins were run on SDS-PAGE and tested by immunoblotting before using in in vitro enzyme activity assay. For antibody production, a His-tagged S-Lap1 fusion protein construct was generated in a pET28b vector (Novagen). The transgenic construct was transformed into Escherichia coli BL21 strain, and expression was induced with 0.1 M IPTG overnight at 18°C. The S-Lap1-His protein was purified with BugBuster Protein Extraction Reagent (Novagen) following the manufacturer's instructions, and then the S-Lap1 protein was purified from a 10% acrylamide gel according to Michalk et al. [Full-length coding DNA sequences of k strain . Bacterik et al.  and used2, 1mM DTT), and then centrifuged at 15000 rcf at 4°C. Equal amounts of bacterially expressed and purified recombinant His-Grsm, His-S-Lap1 and His-S-Lap6 proteins were used in an in vitro aminopeptidase assay. The leucyl aminopeptidase activity was measured from 100 μl of the testis supernatant or purified protein, after adding 100 μM L-Leu-AMC (Sigma-Aldrich), by fluorescence emission at 460 nm (340 nm excitation) using FLUOstar OPTIMA Plate Reader.Testes from 10 males/genotype were dissected and sonicated in 400 μl incubation buffer  equipped with a Morada camera and iTEM software (Olympus).2O2 at RT for 1 minute and washed 3x5 minutes in distilled water, then treated with 0.1% NaHB4 in TBS buffer (pH 5.5) for 10 min, and finally with 50 mM glycin in TBS for 30 minutes. Next, the samples were washed in TBS buffer for 3x5 minutes and blocked for 30 minutes in 10% FBS, 5% dried milk, 1% BSA diluted in TBS. Mouse anti-S-Lap1 (1:30) and rat anti-HA (1:10) (Roche) antibodies were diluted in solutions containing 5% FBS, 2.5% dried milk, 1% BSA in TBS and applied on the sections overnight at 4°C, then washed 3 times with 2% FBS, 1.25% dried milk, 1% BSA in TBS for 5 min. The primary antibodies were detected by incubating the samples with anti-rat IgG (whole molecule)-gold antibody (Sigma-Aldrich) or anti-mouse IgG (whole molecule)-gold antibody (Sigma-Aldrich), where IgG is conjugated with 10 nm gold particles, in a 1:50 dilution at room temperature for 120 minutes. Sections were washed in TBS 3x5 minutes and put in 1% cacodylate buffered glutaraldehyde for 10 minutes to enhance the attachment of gold particles; rinsing in distilled water 3x5 minutes completed the procedure. Sections were evaluated using a JEM-1011 electron microscope (JEOL) equipped with a Morada camera and iTEM software (Olympus).For immunogold labeling, 90 nm sections were treated first with 5% HProtein sequence alignment was made using Clustal Omega (EMBL-EBI). Analysis of intracellular localization signal was made by Predotar  and thenS1 Fig(A) Diagram shows the relative fluorescence intensity measured in the leucyl aminopeptidase assay from the testes of homozygous S-Lap mutants after adding the fluorescent substrate L-Leu-AMC. No significant reduction in aminopeptidase activity is observed in the S-Lap mutants compared to wild-type (WT). Error bars indicate mean +s.e.m., n = 3. (B) Immunoblot of the purified recombinant His-S-Lap1, His-S-Lap6 and His-Grsm proteins using anti-His antibody. (C) An equal amount of His-S-Lap1, His-S-Lap6 and His-Grsm proteins were tested in the leucyl aminopeptidase assay by measuring the relative fluorescence intensity of the fluorescent substrate L-Leu-AMC for 6 hours.(TIF)Click here for additional data file.S2 FigS-Lap1Δ7, S-Lap2MI14597, S-Lap3MB01319, S-Lap4MB11296, S-Lap5Δ14, S-Lap6MI06848, bamGal4>S-Lap7KK105511, bamGal4>S-Lap8KK106866) were visualized by nuclear (Hoechst (blue)) and, mitochondrial (Mitotracker (red)) staining and phase contrast microscopy. Scale bars: 20 μm. The whole testis with elongated cysts was visualized with phase contrast microscopy. Scale bars: 200 μm.Different stages of spermatogenesis  in a whole testis in wild type and S-Lap mutants ((TIF)Click here for additional data file.S3 FigS-Lap1Δ7, S-Lap2MI14597, S-Lap3MB01319, S-Lap4MB11296, S-Lap5Δ14, S-Lap6MI06848, bamGal4>S-Lap7KK105511, bamGal4>S-Lap8KK106866) stained with Axo49 tubulin antibody (green) and nuclei with DAPI (blue). Scale bar: 200 μm. (B) The boxplot shows the length of the Axo49 positive cysts in WT and S-Lap mutants.(A) Confocal micrographs of elongated spermatid cysts of wild-type (WT) and S-Lap mutants ((TIF)Click here for additional data file.S4 FigS-Lap1Δ7, S-Lap2MI14597, S-Lap3MB01319, S-Lap4MB11296, S-Lap5Δ14, S-Lap6MI06848, bamGal4>S-Lap7KK105511, bamGal4>S-Lap8KK106866) show elongated mitochondria stained with Mitotracker (red) and investment cones marked by phalloidin (green) with synchronized and unsynchronized movement. Scale bar: 25 μm.(A) Individualization complexes were counted on phalloidin stained testes samples in S-Lap mutants. n(testis) represents the number of the analyzed testes. (A’) Needle shaped nuclei with individualization complexes, (A”) migrating individualization complexes and (A”‘) disturbed migrating individualization complexes were counted in WT and S-Lap mutants. Statistical significance was determined by Welch two sample t-test. (B) Confocal micrographs of elongated spermatids in wild type (WT)and S-Lap mutants ((TIF)Click here for additional data file.S5 FigS-Lap1Δ7, S-Lap2MI14597, S-Lap3MB01319, S-Lap4MB11296, S-Lap5Δ14, S-Lap6MI06848, bamGal4>S-Lap7KK105511, bamGal4>S-Lap8KK106866). Individualization complexes were stained with phalloidin (red) and nuclei with DAPI (blue). Scale bar: 200 μm. (B) Investment cones are synchronized and mitochondrial DJ-GFP distribution is smooth, while in the S-Lap1Δ7 mutant investment cones are synchronized and DJ-GFP distribution is slightly dashed. In the S-Lap2MI14597 mutant and the S-Lap3MB01319 mutant, the investment cones are dispersed and DJ-GFP distribution is dashed. Scale bar: 50 μm. (C) S-Lap1 antibody (red) decorates weakly the nebenkern in round spermatids (arrows) and the elongating and the elongated mitochondria (arrowhead) both in WT and S-Lap1 mutants. The signal intensity is reduced in the S-Lap1Δ7 testis by using the same exposure time as in the WT. Nuclei were stained with DAPI (blue). Scale bars: 200 μm and 50 μm. (D) Immunoblot of WT and S-Lap1Δ7 testes using S-Lap1 antibody, where one lane represents 1 testis equivalent total lysate. S-Lap1 antibody recognizes the recombinant His-S-Lap1 protein. Note that the His-tagged unprocessed recombinant protein runs higher (~70 kDa) than the processed and untagged endogenous S-Lap1 (~55 kDa).Characterization of the S-Lap1 antibody. (A) Cleaved-Caspase3 (green) is present in the elongated cysts in wild type (WT) and S-Lap mutants ((TIF)Click here for additional data file.S6 FigS-Lap1Δ7 mutant expressing the S-Lap1 genomic rescue construct (S-Lap1resc), stained with phalloidin (red) and the nuclei with DAPI (blue). The seminal vesicle contains individualized sperms . Scale bars: 200 μm  50 μm (B”). (C) Three-dimensional models of S-Lap proteins compared to the bovine leucine aminopeptidase protein model 2EWB. (D) Three-dimensional model alignment of S-Lap1 (red) and S-Lap1-HAint (blue) and the position of the internal 1xHA tag (green).  Cryo cross-sections of wild-type spermatids immuno-labeled with (E) pre-immune serum of S-Lap1 antibody and anti-HA antibody (F) do not show specific labeling after developing with nanogold particles. Brown dashed line borders one elongated spermatid with axoneme (red dashed line) and with major mitochondrial derivative (yellow dashed line) inside. Scale bars: 0.2 μm. Confocal micrographs of elongated cysts stained with mouse pre-immune serum of S-Lap1 antibody (green), and Mitotracker (red) in squash preparation of wild-type testis. Scale bar: 10 μm. (B) Phase contrast and fluorescent images  of the testis of the (TIF)Click here for additional data file.S7 Fig(A) Analysis of protein accumulation in the 50-60 kDa molecular weight region of SDS-resistant pellet fraction of sperm extract by mass spectrometry. (B) S-Lap proteins contain an N-terminal mitochondrial target sequence with high probability. The MPP  cleavage sites were predicted using the online software MitoFates. (C) Sequence similarity between S-Lap proteins. Alignment analysis was made by Clustal Omega software.(TIF)Click here for additional data file.S8 Fig(A) The sequences used for generating sgRNA lines and (B) the primers used for cloning transgenic constructs.(TIF)Click here for additional data file.


Scientific Reports 10.1038/s41598-018-38399-1, published online 04 February 2019Correction to: This Article contains an error in Figure 8f, where the incorrect image was used for the telmisartan treatment (1 mg/kg) in brown fat tissue. The correct Figure


Compared to wild-type BMDCs, the Fas-deficient BMDCs had increased ERK activity and decreased IL-12 production upon HDM stimulation. Inhibition of ERK activity could largely increase IL-12 production, consequently restored the increased Th2 cytokine expression of OT-II CD4+ T cells activated by Fas-deficient BMDCs. Thus, our results uncover an important role of DC-specific Fas signaling in Th2 differentiation and allergic inflammation, and modulation of Fas signaling in DCs may offer a useful strategy for the treatment of allergic inflammatory diseases.Fas–Fas ligand (FasL) signaling plays an important role in the development of allergic inflammation, but the cellular and molecular mechanisms are still not well known. By using the bone marrow-derived dendritic cell (BMDC) transfer-induced pulmonary inflammation model, we found that house dust mite (HDM)-stimulated FAS-deficient BMDCs induced higher Th2-mediated allergic inflammation, associated with increased mucus production and eosinophilic inflammation. Moreover, FAS-deficient BMDCs promoted Th2 cell differentiation upon HDM stimulation  Allergic inflammation has been generally considered as a T helper (Th) 2-mediated chronic immune response . Th2 celFas  signaling is widely considered to mediate apoptosis upon binding to its ligand  or its agonist antibody . In an oin vivo. Fas-deficient BMDCs promoted the production of Th2-related cytokines such as IL-4 and IL-13. Further mechanistic study showed that DCs directed Th2 differentiation by modulating the Fas–ERK–IL-12 axis. Collectively, our results identify an important signaling mechanism of DC-mediated Th2 responses and modulation of Fas signaling in DCs might offer a useful strategy for the treatment of eosinophilic lung inflammatory diseases.In the present study, we used the well-established model by adoptively transferring HDM-pulsed BMDCs to recipient mice to explore the role of FAS signaling in DCs in pulmonary inflammation. We found that Fas deficiency in DCs led to increased mucus production, eosinophilic inflammation and Th2 response Tnfrsf6lpr mice were from The Jackson Laboratory. C57BL/6 mice were from Shanghai SLAC Laboratory Animal Center . All mice were kept in a specific pathogen-free (SPF) barrier facility maintained by Shanghai Jiao Tong University School of Medicine. All the experimental mice were used at 6–10 weeks. Animal protocols were approved by Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.B6.MRL-+ BMDCs was >80%.BMDCs were cultured as previously described . Briefly6 HDM-pulsed BMDCs were administered intravenously into naïve C57BL/6 recipients. Recipients transferred with un-pulsed wild-type or Fas-deficient BMDCs as control. On day 10–12, all the recipients were lightly anesthetized and challenged intranasally with 10 μg HDM in 40 μl PBS and the mice were sacrificed for analysis on day 13.In BMDC adoptive transfer experiment, wild-type or Fas-deficient BMDCs were pulsed with 50 μg/ml HDM  for 12 h, washed, and then 1 × 10+T cell proliferation was detected by anti-Ki-67 staining according to the manufacturer's instructions (eBioscience) 3 days after co-culture. Labeling OTII CD4+ T cells with CFSE , cell proliferation was detected by flow cytometry 4 days after co-culture. For cell apoptosis analysis, cells were stained with CaspACE™ FITC-VAD-FMK in situ Marker (Promega). The samples were acquired on a FACSCantoII (BD) or LSRFortessaTM X-20 (BD) and analyzed with FlowJo software (Treestar).Anti-mouse CD11c (N418), MHC-II (M5/114.15.2), CD11b (M1/70), Ly6G (RB6-8C5), siglec F (E50-2440), CD4 (RM4-5), TCRβ (H57-597), IL-4 (11B11), IL-13 (eBio13A), IL-17A (eBio17B7), CD25 (PC61.5), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), Ki-67 (SolA15), IL-12p40 (C17.8), CD45 (30-F11), CD178 (MFL3) antibodies were obtained from eBioscience. Anti-mouse IL-5 (TRFK5) was obtained from BD Biosciences. Anti-mouse CCR3 (J073E5) was obtained from Biolegend. For surface staining, cells were stained with antibodies in PBS containing 1% FCS (Hyclone) on ice for 30 min. For intracellular staining, cells were stimulated with PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich) for 5 h in the presence of Golgistop (BD Biosciences) before being stained according to the manufacturer's instructions (eBioscience). CD4For BALF collection, lung tissues were lavaged with 1 ml cold PBS for 3 times and the supernatant was collected. Lung mononuclear cells were prepared as previously described . BrieflyLeft lobe of lung tissues were removed from mice after BALF collection, fixed with 4% paraformaldehyde (PFA) at room temperature for 24 h and embedded in paraffin, cut into 5-μm sections for hematoxylin and eosin (HE) or periodic acid-Schiff (PAS) staining. The lung inflammation was blindly quantified using HE-stained sections according to the criteria previously published . The quaHprt. The primers shown below were from Primerbank: Hprt, forward primer: TCAGTCAACGGGGGACATAAA, reverse primer: GGGGCTGTACTGCTTAACCAG; Il4, forward primer: GGTCTCAACCC CCAGCTAGT, reverse primer: GCCGATGATCTCTCTCAAGTGAT; Il17a, forward primer: TCAGCGTGTCCAAACACTGAG, reverse primer: CGCCAAGGGAG TTAAAGACTT; Ifng, forward primer: GCCACGGCACAGTCATTGA, reverse primer: TGCTGATGGCCTGATTGTCTT; Il6, forward primer: CTGCAAGAGACTTCCATCCAG, reverse primer: AGTGGTATAGACAGGTATGTTGG; Il12p35, forward primer: CAATCACGCTACCTCCTCTTT, reverse primer: CAGCAGTGCAGGAATAATGTTTC; Il12p40, forward primer: GTCCTCAGAAGCTAACCATCTC, reverse primer: CCAGAGC CTATGACTCCATGTC; Il10, forward primer: CTTACTGACTGGCATGAGGATCA, reverse prime: GCAGCTCTAGGAGCATGTGG. The other primers were used as described, such as Il5, Il13 . Total RNAs of cells were isolated using RNeasy mini Kit (QIAGEN) according to the manufacturer's instructions. 1 μg total RNA was used for reverse transcription with PrimeScript RT Master Mix (TAKARA) according to the manufacturer's instructions in a total volume of 20 μl. qPCR was carried out with SYBR Green PCR Master Mix (Applied Biosystems) in a Vii7 Real-Time PCR system (Applied Biosystems). mRNA expression of genes was normalized to 3  were mixed at a ratio of 1:10 in the presence of OVA323−339 peptide and HDM, and then the CD4+ T cells were harvested at 48 h for mRNA analysis or supernatant was harvested at 72 h for ELISA. For cytokine treatment, cultures were supplemented with 1 ng/ml IL-12p70 (R&D).BMDCs were stimulated with HDM in the presence or absence of Fas agonistic antibody Jo2  or Fas antagonistic antibody kp7-6  for 5 h for RNA analysis. For drug inhibitor treatments, cells were incubated with vehicle (DMSO) or U0126 (10 μM)  for 0.5–1 h before adding other stimuli. For BMDC–T cell co-culture, BMDCs and flow cytometry-sorted naïve OT-II CD4Concentrations of IL-4 and IL-13 were measured by ELISA according to the manufacturer's instructions . Read the OD values at 450 nm on the MultiSKAN GO microplate reader (Thermo). Immunoblot analysis was performed as described  with antt-tests or ANOVA using GraphPad Prism software (version 5.0). P < 0.05 was considered significant. Results represent means ± SEM.All statistical analyses were performed by unpaired Student's +Siglec FintCCR3+CD62L−) and resident eosinophils (rEos) (CD45+Siglec FintCCR3+CD62L+) in lung tissues  value of VAD+CD4+ T cell in lung tissues and mediastinal lymph nodes (mLN) had no differences between the recipients transferred with HDM-pulsed wild-type BMDCs and those transferred with Fas-deficient BMDCs , Il5 (encoding IL-5), and Il13 (encoding IL-13), but comparable expression of Ifng (encoding IFNγ) and Il17a (encoding IL-17A) in lung tissues than those transferred with HDM-pulsed wild-type BMDCs (Figure + T cells was observed in lung tissues of the two HDM-pulsed groups (Figure +, IL-5+, and IL-13+CD4+ T cells in lung tissues than those transferred with wild-type BMDCs, along with higher cell number of IL-4+, IL-5+, and IL-13+CD4+ T cells (Figures +CD4+ T cells were similar in the two groups (Supplementary Figures +, IL-5+, and IL-13+CD4+ T cells had no difference between the recipients transferred with un-pulsed wild-type and Fas-deficient BMDCs (Supplementary Figures in vivo.Given that there was no difference in the proliferation and apoptosis of CD4s Figure . Althougs Figure , intrace Figures , whereasin vivo is due to the direct interaction between DCs and T cells, we co-cultured wild-type or Fas-deficient BMDCs with naïve OT-II CD4+ T cells in the presence of OVA323−339 peptide with or without HDM. The polarization of CD4+ T cells was determined by qPCR or ELISA, respectively. OT-II CD4+ T cells activated with HDM-stimulated Fas-deficient BMDCs had higher IL-4 and IL-13 expression both in mRNA and in protein levels than those stimulated with wild-type BMDCs (Figures Il9 and Il10 was comparable (Figure + T cells activated by HDM-pulsed both wild-type and Fas-deficient BMDCs were comparable (Figures in vitro.To determine whether the increased Th2 response  Figures . GATA3, e Figure . The act Figures –E. ColleCd86, Tslp, Il10, Il6, and Tnfsf4, which had been reported to regulate Th2 polarization, had no difference compared to control group (Figure Il6 and Tnfsf4, while Fas agonistic antibody Jo2 did not affect the expression of these two genes (Figure Il6 and Tnfsf4 expression in DCs upon HDM stimulation. IL-12 has been reported to affect Th2 differentiation (Il12p35 and Il12p40 in wild-type BMDCs without HDM stimulation (Figure Il12p35 and Il12p40 was significantly increased compared with non-HDM stimulation, and Fas agonistic antibody Jo2 could further enhance the expression of Il12p35 and Il12p40 in mRNA level and IL-12p70 in protein level (Figures Il12p40 in mRNA level and IL-12p70 in protein level than those of BMDCs stimulated with HDM alone, but the expression of Il12p35 had no difference between HDM and HDM plus kp7-6 stimulated BMDCs (Figures Il12p35 and Il12p40 compared to wild-type BMDCs upon HDM stimulation in the presence of Fas agonistic antibody Jo2 (Figure + T cells activated by Fas-deficient BMDCs. The expression of IFNγ and IL-17 was comparable between wild-type and Fas-deficient BMDC activated T cells (Figure Next we explored the molecular mechanism by which Fas signaling in BMDCs to shape Th2 differentiation upon HDM stimulation. We stimulated wild-type BMDCs with Fas agonistic antibody Jo2, the expression of p Figure . HDM stis Figure , indicatntiation . Fas agon Figure . Upon HD Figures . Given t Figures . Fas-def2 Figure . We nexts Figure . AltogetWe next examined the downstream signaling of Fas involved in the regulation of IL-12 expression by analyzing the activation of p38, JNK, and ERK in BMDCs. We found that the phosphorylation of ERK was decreased in wild-type BMDCs treated with Fas agonistic antibody Jo2 and HDM compared to those treated with HDM alone Figure . AccordiNumerous studies have reported that administration of allergen-pulsed DCs is sufficient to induce airway inflammation by polarizing Th2 responses , 29–31. DCs constitutively express non-canonical costimulatory molecule Fas , which i+ T cells has a key role in Th2 cell polarization (Neospora caninum infected peritoneal macrophages (ERK signaling has been shown to play important roles in Fas-mediated non-apoptotic function. Ligation of Fas agonistic antibody Jo2 with Fas on DCs can promote the activation of ERK and subsequent IL-1β secretion . In the rization . Blockinrophages . In controphages . In thisin vivo and cellular experiments and contributed to manuscript writing. RH performed the in vitro and cellular experiments. JM contributed mouse models. CC contributed to animal colony management. BZ contributed to western blot analysis. HL and JY provided reagents. GH designed experiments, analyzed the data, wrote the manuscript, and provided overall directions.MH designed and performed the 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.