In vivo treatment of MS-444 inhibited HuR cytoplasmic localization and decreased COX-2 expression in tumors. These findings provide evidence that therapeutic strategies to target HuR in CRC warrant further investigation in an effort to move this approach to the clinic.Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality. Observed during CRC tumorigenesis is loss of post-transcriptional regulation of tumor-promoting genes such as COX-2, TNFα and VEGF. Overexpression of the RNA-binding protein HuR (ELAVL1) occurs during colon tumorigenesis and is abnormally present within the cytoplasm, where it post-transcriptionally regulates genes through its interaction with 3′UTR AU-rich elements (AREs). Here, we examine the therapeutic potential of targeting HuR using MS-444, a small molecule HuR inhibitor. Treatment of CRC cells with MS-444 resulted in growth inhibition and increased apoptotic gene expression, while similar treatment doses in non-transformed intestinal cells had no appreciable effects. Mechanistically, MS-444 disrupted HuR cytoplasmic trafficking and released ARE-mRNAs for localization to P-bodies, but did not affect total HuR expression levels. This resulted in MS-444-mediated inhibition of COX-2 and other ARE-mRNA expression levels. Importantly, MS-444 was well tolerated and inhibited xenograft CRC tumor growth through enhanced apoptosis and decreased angiogenesis upon intraperitoneal administration.  The lifetime risk of developing colorectal (CRC) cancer is ~5% for both men and women in the USA . VariousDrosophila embryonic-lethal abnormal vision (ELAV) proteins [A key factor that mediates ARE-mRNA stability is HuR (ELAVL1), which is a member of a family of proteins analogous to the proteins . HuR hasproteins –10. In nproteins , 11, 12.Previous work has identified HuR as a central protein in the process of tumorigenesis. HuR has been shown to increase cell division by enhancing the stability of several mRNAs that regulate growth and proliferation, such as cyclin A, cyclin B1 and c-fos , 14. AddIn light of its central role in tumorigenesis and its differential expression between normal and cancerous tissues, HuR has emerged as an attractive therapeutic target for cancer –20. Effoin vitro and in vivo. Importantly, MS-444 efficiently inhibited CRC tumor growth in pre-clinical models, thereby underscoring HuR inhibition as a viable and cancer cell-specific therapeutic strategy.In this report, we demonstrate that cytoplasmic HuR localization sensitizes colon cancer cells to the growth-inhibitory effects of MS-444, while non-transformed intestinal epithelial cells are refractory to these effects. Additionally, we demonstrate that HuR inhibition by MS-444 results in inhibition of COX-2 expression both Micromonospora and originally characterized as myosin light chain (MLC) kinase inhibitor [146 and Met117 with bond lengths of 2.7 Å and 2.5 Å, respectively   , 29, 30 4 Figure . Contras) Figure . Additio) Figure .Previous studies have demonstrated HuR overexpression to be anti-apoptotic through its influence on the expression of multiple pro-survival mRNAs , 36. To Rapid ARE-mRNA decay occurs primarily in the cytoplasm, however the presence of cytoplasmic HuR in CRC cells impedes this process , 37, 38.V12, and when cultured in the presence of IPTG for 24 hr to induce oncogenic Ras, undergo epithelial-to-mesenchymal transition (EMT) [50 of 2.33 μM, as compared to 31.93 μM in non-transformed cells  . Shown is Figure . Similars Figure , further50 of 6.75 μM  was performed in HCA-7 cells treated with 10 μM MS-444 for 6 hr and immunoprecipitation of cytoplasmic lysates was done using an antibody against HuR. The association of COX-2 mRNA with HuR was assayed by qPCR of COX-2 mRNA in immunoprecipitates. As shown in Figure In non-transformed intestinal epithelial cells, COX-2 and ARE-mRNAs are degraded in discrete cytoplasmic RNA granules known as processing bodies (P-bodies) . To detePrior examination of MS-444 acute toxicity in rats showed no lethality at concentrations of 100 mg/kg by intraperitoneal (IP) administration . To detein vivo, mice bearing HCT116 cell xenografts received IP injections of MS-444 (25 mg/kg bw) or vehicle every 48 hr. Over the experiment course, mice did not display any adverse effects and maintained similar weights  and then treated intraperitoneally with MS-444 (25 mg/kg bw) every 72 hr for 15 days followed by a 7-day period during which they received daily injections of the same dose  silence expression levels , 2) disrBased on HuR's ability to support an anti-apoptotic network through post-transcriptional mechanisms , 35, we in vivo through enhanced apoptosis and decreased angiogenesis, implicate that selective targeting of these pathways in combination with MS-444 may improve the limited inhibition on tumor growth by MS-444 as a monotherapy. Further efforts to evaluate these findings in CRC models as a means to identify clinically available targeted and cytotoxic therapies that would synergize with HuR targeted therapy are in progress. These efforts, along with other modes of HuR inhibition  that induce HuR cytoplasmic localization in pancreatic cancer cells , 59–61. delivery ), being V12 cDNA. Conditionally immortalized colonocytes YAMC and YAMC-Ras (YAMC cells overexpressing the v-Ha-Ras oncogene) cells were maintained under permissive conditions as described [−1cm−1 at 328 nm.Human colorectal cancer cells SW480, HT-29, HCT116, and RKO were obtained from the American Type Culture Collection (ATCC); HCA7 cells were provided by S. Kirkland . Cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone). Intestinal epithelial cells lines RIE-1, RIE-iRas, YAMC, and YAMC-Ras were provided by R. D. Beauchamp . RIE-iRas cells were cultured as described  using 5 escribed  at 33°C Molecular docking was performed using the X-ray crystal structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form (PDB ID: 4EGL) . MS-444 50) was derived and represented as a mean of 4 independent experiments ± standard error of the mean (SEM).Cells were seeded at subconfluent levels (<50% confluence) in 96-well tissue culture plates and treated with increasing concentrations of MS-444  for 48 h6 cells were resuspended in Annexin V binding buffer and incubated with 5 μL Annexin V-PE and 5 μL propidium iodide for 15 minutes in the dark. Cells were subjected to flow cytometry using the BD Accuri C6 flow cytometer. The collected data was subjected to compensation analysis by adjusting the gating and virtual voltage in the BD Accuri C6 software and graphically represented as means of 3 independent experiments. Cells treated with 5 μg/ml cycloheximide (Chx) (Sigma-Aldrich) and 10 ng/ml TNF-α (R&D Systems) for 8 hr were used as positive controls.HCT116 and RIE-1 cells treated with 10 μM MS-444 for 48 hours were subjected to Annexin V staining and flow cytometry per manufacturer's protocol (ThermoFisher-Molecular Probes). 1×10Western blots were performed as described  using ant) using non-treated cells and analyzed by the ΔΔCt method.Total RNA was extracted using Trizol reagent (Invitrogen). cDNA synthesis was performed using 1 μg of total RNA in combination with oligo(dT) and Improm-II reverse transcriptase (Promega). qPCR analysis was performed as described  using th2, 0.05% NP-40) and incubated with equal amounts (250 μg) of cytoplasmic lysates obtained from cells lysed in 200 μl polysome lysis buffer  containing 100 U/ml RNase inhibitor (Ambion) and protease inhibitor cocktail (Sigma-Aldrich) and incubated overnight at 4°C with HuR- or control-IgG coated beads. Reactions were washed 5X with NT2 buffer and total RNA was isolated from immunoprecipitates using 1 mL TRIzol per IP reaction and then used for cDNA synthesis. Analysis of COX-2 (PTGS2) mRNA was done by qPCR as described above.Immunoprecipitation (IP) of ribonucleoprotein complexes (RNP-IP) was done as described  using HCLuciferase reporter constructs containing the COX-2 3′UTR (Luc+COX-2 3′UTR) or control (LucΔ3′UTR) were transfected as previously described . Cells wThe MS2-based plasmids pcDNA-MS2-YFP and pMS2-COX-2 3′UTR used for fluorescent visualization of RNA, and the P-body marker mCherry-tagged Dcp1a (pmCherry-Dcp1a) have been previously described , 40. TheCells grown on coverslips were washed twice with PBS containing 10 mM glycine, fixed in 2% paraformaldehyde for 15 min at RT, and permeabilized with 0.02% Triton X-100 (Sigma-Aldrich) in PBS. Cells were blocked with 5% normal goat serum in PBS containing 1% IgG-free BSA (Jackson Immunoresearch) and incubated with primary antibody overnight at 4°C diluted in blocking solution. HuR was detected with anti-HuR monoclonal primary antibody  for 1 hr at RT. Secondary antibody incubation was done for 1 hr at RT using FITC-conjugated anti-mouse IgG (1:100 dilution) or Alexa488-conjugated anti-mouse IgG . Cells were counter-stained with DAPI to visualize nuclei. Fluorescence microscopy and image analysis was accomplished as described , 61. Det6 cells) and HCA-7 (2.5 × 106) cells resuspended in PBS were injected into the dorsal subcutaneous tissue. Mice (n=5 per group) received intraperitoneal (IP) injections of MS-444 (25 mg/kg) dissolved in PBS/5% N-Methyl Pyrrolidine (NMP) (Sigma-Aldrich) or vehicle control every 48 hr. Tumor growth was assayed as described [Athymic nude (Nu/Nu) mice were purchased from Harlan Laboratories and maintained under sterile conditions in cage micro-isolators according to approved IACUC guidelines. HCT116  tumors sectioned at 4 μm with monoclonal anti-HuR antibody  at 1:1250, polyclonal anti-cleaved caspase 3 (Asp175) antibody  at 1:1000, and polyclonal anti-COX-2  at 1:400 dilutions. Staining and immunoreactivity scoring was performed as described previously .Tumor microvessels were detected by IHC ofFFPE tumors using a polyclonal antibody to CD31  at a dilution of 1:500 for 1 hr at RT in primary antibody diluent (Dako). After washing in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), slides were incubated with biotinylated anti-rabbit labeled polymer (Dako). IHC was visualized using DAB peroxidase substrate kit (Dako) and counterstained with hematoxylin (Sigma-Aldrich). The area of tumor microvessels was quantitatively measured using the ImagePro Plus 4.5 software (Media Cybernetics) as previously described . Tumor s3) were observed, mice were treated with MS-444 or vehicle control as indicated. COX-2 protein expression was visualized ex vivo by IP injection of 2 mg/kg of Fluorocoxib  for 3 hr. Mice were euthanized and excised tumors were imaged in an IVIS platform  with the following settings: 570 excitation, 620 emission, F-1 Stop, epifluorescence. Following visualization, the tumors were snap-frozen and processed for RNA and protein analysis.Athymic nude (Nu/Nu) mice bearing HCA-7 xenografts were established as described above. When palpable tumors . The data are expressed as the mean of three independent experiments ± S.E.M. Student's


Cuc B induced autophagy as evidenced by monodansylcadaverine (MDC) staining, increased expression of LC3II, phosphorylated ULK1, and decreased expression of phosphorylated AKT, mTOR. Cuc B induced apoptosis mediated by Bcl-2 family proteins and caspase activation. Furthermore, Cuc B induced ROS formation, which was inhibited by N-acetyl-L-cysteine (NAC). NAC pretreatment dramatically reversed Cuc B-induced DNA damage, autophagy, and apoptosis. Cuc B-induced apoptosis was reversed by NAC but enhanced by 3-methyladenine (3-MA), chloroquine (CQ), and silencing phosphatase and tensin homolog (PTEN). 3-MA and CQ showed no effect on Cuc B-induced DNA damage. In addition, Cuc B increased PTEN phosphorylation and silence PTEN restored Cuc B-induced autophagic protein expressions without affecting DNA damage. In summary, Cuc B induced DNA damage, apoptosis, and protective autophagy mediated by ROS. PTEN activation in response to DNA damage bridged DNA damage and prosurvival autophagy.Cucurbitacin B (Cuc B), a natural product, induced both protective autophagy and DNA damage mediated by ROS while the detailed mechanisms remain unclear. This study explored the mechanism of Cuc B-induced DNA damage and autophagy. Cuc B decreased cell viability in concentration- and time-dependent manners. Cuc B caused long comet tails and increased expression of  Programmed cell death (PCD), a process carried out in a regulated manner, ubiquitously occurs throughout most multicellular organisms' lifespan. To date, three major types of PCD, distinct both morphologically and biochemically, have been established: apoptosis (type I cell death), autophagic cell death (type II), and regulated necrosis (type III) –3. The fAccumulated evidence showed that though apoptosis and autophagy were executed through distinct signaling pathways, overlapping signals were engaged in response to specific stimuli . This crCucurbitacin B (Cuc B), a natural tetracyclic triterpenoid, is abundant in many Cucurbitaceae species . Cuc B iγ-H2AX, PTEN, phosphorylated PTEN (p-PTEN (Ser380/Thr382/Thr383)), AKT, phosphorylated AKT (p-AKT (Ser473)), ULK1, phosphorylated ULK1 (p-ULK1 (Ser317)), mTOR, phosphorylated mTOR (p-mTOR (Ser2448)), p62, LC3, Bcl-2, Bik, Bak, cleaved-PARP, cleaved-caspase 7, and cleaved-caspase 9 and secondary antibodies were bought from Cell Signal Technology . KU55933 were obtained from Selleck . Caffeine, monodansylcadaverine (MDC), 3-methyladenine (3-MA), and 5-(6)-carboxy-2′,7′-dichlor-odihydrofluorescein diacetate (DCFH2-DA) were purchased from Sigma . N-Acetyl-L-cysteine (NAC) and chloroquine (CQ) were purchased from Beyotime . Protein phosphatase inhibitor cocktail and propidium iodide (PI) were from Gibco/Thermo Fisher Scientific .Cuc B (>98%) purchased from Chengdu Herbpurify Co., Ltd. , was dissolved in dimethyl sulfoxide (DMSO) to make a 100 mM stock solution and was freshly diluted to the desired concentration before use. Primary antibodies for GAPDH, ATM, phosphorylated ATM (p-ATM (Ser1981)), ATR, phosphorylated ATR (p-ATR (Ser428)), Chk1, phosphorylated Chk1 (p-Chk1 (Ser345)), Chk2, phosphorylated Chk2 (p-Chk2 (Thr68)), 2 at 37°C in RPMI Medium 1640 (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen).Hepatocellular carcinoma BEL-7402 cells gifted by Professor Jian Ding from the Shanghai Institute of Materia Medica  were maintained in a monolayer culture in 95% air and 5% COμM) or transfected with PTEN siRNA and then cotreated with Cuc B.Cells seeded in 96-well plates for overnight and then treated with or without Cuc B (0–100 nM) for 0–48 h and the cell viabilities were determined by MTT assay. To elucidate the role of ROS, autophagy, DNA damage, and PTEN in Cuc B-induced cell death, cells were pretreated with NAC (2.5 mM), 3-MA (2.5 mM), and CQ  according to the manufacturer's instructions.Cells seeded in 6-well plates were treated with or without Cuc B (5–20 nM) and the colony formation assay was performed as in our previous report .Comet assay was performed as in previous report with minor revisions . Brieflyμg) were loaded and electrophoresed on SDS-PAGE gels and then transferred to PVDF membrane (Thermo Scientific). The membranes were firstly blocked with 5% nonfat dry milk for 2 h at room temperature and then incubated with primary antibodies (1 : 3000) overnight at 4°C. Then HRP-linked secondary antibodies (1 : 5000) were incubated for 4 h at room temperature. The bands were visualized with the ChemiDoc™ MP Imaging System (Bio-Rad).Treated cells were washed with PBS twice and then harvested using ice-cold RIPA lysis buffer containing protease inhibitor PMSF (Gibco) and protein phosphatase inhibitor cocktail (Gibco). The lysates were centrifuged at 12,500 g for 20 min at 4°C and the supernatant fractions were collected. Protein concentrations were measured with BCA Protein Assay Kit (Gibco). After denaturation at 95°C for 10 min, equivalent aliquots of protein samples .The condensation of DNA was detected by Hoechst 33342 staining as in our previous report .p < 0.05 is considered statistically significant.Data were expressed as the means ± SEM from at least three separate experiments performed in triplicate. The differences between groups were analyzed using Prism 5.0  and Compared with control, morphological alterations were observed in Cuc B-treated cells, especially in 100 nM Cuc B-treated cells . MTT assγ-H2AX expression in dose- (The comet assay showed that significant long tails were observed in Cuc B-treated cells  indicatiin dose-  and timein dose- . Furtherin dose- . In addiin dose-  and caffin dose- .Compared with the control group, Cuc B-treated cells showed dramatic increase of intensive green fluorescence in MDC staining suggesting the accumulation of autophagic vacuoles . Cuc B tCuc B induced apoptosis in a dose-dependent manner . FurtherCuc B significantly induced ROS formation in a dose-dependent manner after 1 h treatment , which wγ-H2AX, and phosphorylation of ATM and ATR and Chk1 and Chk2 were significantly decreased by NAC pretreatment (The roles of ROS in Cuc B-induced DNA damage, autophagy, and apoptosis were further examined. Cuc B-induced expression of DNA damage response related proteins, reatment . Furtherreatment . NAC aloreatment .γ-H2AX expression (Figures Since both DNA damage and autophagy were initiated by Cuc B, their relationship was clarified by applying DNA damage response inhibitors KU55933 and caffeine and autophagy inhibitors 3-MA and CQ. KU55933 and caffeine reversed Cuc B-induced decrease of p-mTOR and p-AKT Figures . They al Figures . In addi Figures .Phosphorylation of PTEN was obviously upregulated by Cuc B in a dose-dependent manner . IncreasTo dissect the role of PTEN in Cuc B-induced DNA damage, comet assay was performed. PTEN silence showed no effect on the lengths of comet tails  and p-ATWe previously reported that Cuc B induced ROS-mediated DNA damage in A549 cells . The maiγ-H2AX expression suggesting that it induced DNA damage in BEL-7402 cells. The key regulators in response to DNA damage are ATM and ATR kinases, which activated Chk1 and Chk2 [Cuc B-induced cell death was well established in many cell lines. The MTT and colony formation results showed that Cuc B decreased cell viability and inhibited proliferation at nano-mol levels. Increased LDH release suggested that Cuc B might affect cell membrane. We previously reported that Cuc B induced DNA damage in A549 , K562 2, and MCFand Chk2 , and ATMand Chk2 . Thus, CCuc B-induced autophagy was observed in Jurkat  and MCF-Induction of apoptosis by Cuc B was documented. Cuc B induced apoptosis in BEL-7402 cells as evidenced by Annexin V/PI double staining and the Hoechst 33342 staining. Furthermore, Cuc B increased the proapoptotic proteins Bak and Bik expression. However, the antiapoptotic protein Bcl-2 was slightly decreased by Cuc B. Thus, Cuc B-induced apoptosis might be mainly through the upregulation of proapoptotic Bcl-2 family proteins. In addition, the increased cleavage of caspase-7, caspase-9, and PARP revealed that apoptosis was caspase-dependent.Cuc B-induced ROS played important roles in DNA damage, apoptosis, and autophagy , 27, 29.PTEN, a tumor suppressor gene, has been demonstrated to play a critical role in DNA damage repair and DNA damage response . It alsoIn summary, as depicted in


Arcobacter from saline fluids produced from hydraulically fractured shale gas wells in the Marcellus and Utica formations. These genomes provide insight into microbial sulfur cycles occurring in a high-salt deep terrestrial shale environment.Genome sequences were obtained for two isolates of the genus  Arcobacter marinus UTICA-S4D1 and MARC-MIP3H16 were isolated from highly saline  produced fluids from two distinct hydraulically fractured shale gas wells in the Utica-Point Pleasant (Ohio) and Marcellus (West Virginia) formations, respectively. Arcobacter, a member of the class Epsilonproteobacteria, is a Gram-negative non-spore-forming curved rod found in a wide range of food, clinical, and marine environments  and 70 days (Marcellus) after flowback began under a low-oxygen environment on Difco Marine agar 2216 at 5% NaCl held at 30°C. Genomic DNA was isolated using a Qiagen DNeasy kit  and sequenced at the Joint Genome Institute, Walnut Creek, CA, USA. Genome assemblies were constructed from Illumina MiSeq data (SPAdes version 3.6.2) and annotated using the Integrated Microbial Genomes platform (Pipeline version 4.12.1). The assemblies generated 69 and 70 contigs for UTICA-S4D1 and MARC-MIP3H16, respectively. UTICA-S4D1 and MARC-MIP3H16 have 2,779 and 2,845 protein-coding genes, with G+C contents of 27.12% and 27.18%, respectively. A ribosomal protein S3 comparison indicated that these two Arcobacter strains are highly similar, with 98.6% average nucleotide identity (ANI).These Arcobacter strains are facultative aerobes capable of utilizing amino acids, propionate, and tricarboxylic acid (TCA) cycle intermediates, but not carbohydrates, for carbon and energy. Additionally, each genome held the potential for autotrophic carbon fixation (napAB) and nitroalkanes (nmo) to ammonia (nrfAH) for energy generation. Both Arcobacter strains contained genes for sulfur oxidation from thiosulfate (soxABCD and soxYZ), tetrathionate (ttr), and sulfide (sqr), which produce corrosive agents commonly detected in shale-produced fluids  numbers 2703719342 and 2700989666, respectively.These whole-genome sequences for MARC-MIP3H16 and UTICA-S4D1 have been deposited in ENA under the accession numbers


Similar ASA-induced effects in-vitro were seen in human melanoma and nasopharyngeal carcinoma cells positive or negative in PAF-R. Mechanistically, the ASA-induced decrease in cell survival and increase in apoptosis were significantly blocked by prostaglandin F2 alpha (PGF2α) agonists. Importantly, PCR array and qRT-PCR analysis of B16-tumors revealed significant downregulation of sry-related high-mobility-box-2 (SOX2) oncogene by ASA treatment. Interestingly, modulation of SOX2 expression by PGF2α agonists and upregulation by fibroblast growth factor 1 (FGF-1) rescued melanoma cells from ASA-induced decreased survival and increased apoptosis. Moreover, PGF2α-receptor antagonist, AL8810 mimics ASA-induced decreased melanoma cells survival which was significantly blocked by PGF2α and FGF-1. These findings indicate that ASA inhibits the growth of aggressive melanoma via SOX2-dependent-PAF-R-indepedent pathway.Acquired resistance to standard therapies remains a serious challenge, requiring novel therapeutic approaches that incorporate potential factors involved in tumor resistance. As cancers including melanoma express inflammatory cyclooxygenases generating prostaglandins implicated in tumor growth, we investigated mechanism of anti-inflammatory drug, acetylsalicylic acid (ASA) which has been shown to inhibit various tumor types, however, its effects against highly aggressive melanoma model are unclear. Given our reports that an activation of platelet-activating factor-receptor (PAF-R) augments the growth and impede efficacies of therapeutic agents in experimental melanoma, we also sought to determine if PAF-R mediates anti-melanoma activity of ASA. The current studies using stably PAF-R-positive (B16-PAFR) and negative (B16-MSCV) murine melanoma cells and PAF-R-expressing and deficient mice, demonstrate that ASA inhibits the  Malignant melanoma is the most aggressive type of skin cancer that contributes about 80% of skin-cancer-related deaths leading to over 9000 annual mortality in the United States . MelanomOur previous studies have shown that an activation of platelet-activating factor-receptor (PAF-R), a seven transmembrane G-protein coupled receptor mediates pro-oxidative stressors-induced acute pro-inflammatory and delayed systemic immunosuppressive responses –23. The Most importantly, malignancies including melanoma express COX-2 and, inhibition of COX-2 or its generated PGs have been shown to exert promising anti-tumor effects against melanoma –27. ProsNotably, aspirin, an acetylsalicylic acid (ASA) has classically been used as an anti-inflammatory drug due to its ability to inhibit COX enzymes resulting in decreased synthesis of PGs, and thus, possesses chemopreventive activities against a variety of cancers –36. WhilAmong various factors involved in inducing tumor resistance or anti-apoptotic responses to standard therapies against cancers, sry-related high-mobility box -2 (SOX-2) gene has emerged as an important oncogene which affects tumor cell behavior including proliferation, apoptosis and resistance to standard therapies –47. Whilin-vitro and in-vivo growth B16F10 melanoma tumors via SOX2-dependent-PAF-R-independent pathway.As ASA targets both COX-dependent and independent pathways –51, the 50 of 5mM at 72 hours as measured via a quantitative sulforhodamine (SRB) assay [50 dose of ASA at 72 hour time point and demonstrated that ASA-mediated inhibition of these melanoma cell survival was due to the induction of apoptosis as measured by immunoblotting for cleaved caspase 3, luminescence and fluorescence-based caspase-3/7 activity  assay . Our nexy Figure . Our nexy Figure . Similary Figure  generatein-vivo melanoma tumor growth with the focus of investigating the role of host-PAF-R. To that end, we first performed pilot studies to determine the optimal dose of ASA that inhibits melanoma tumor growth. Our pilot studies in WT mice (n=5/group) demonstrate that supplementation of ASA  in drinking water inhibits B16-MSCV tumor growth compared to vehicle (0.2% DMSO) in a dose-dependent manner without exerting deleterious effects -PAF-R mediates augmentation of melanoma tumor growth . Our nex effects . The gre effects . To furt effects . These sin-vitro studies used B16-MSCV cells with ASA at IC50 dose of 5mM at 72 hour. To that end, we performed initial studies to test various working doses of PGE2 and PGF2α which by itself do not affect the cell viability (data not shown) and chosen 1.0 μg/ml dose. Next, B16-MSCV cells were cultured with PGE2 and PGF2α at 1.0 μg/ml concentration and cultured for 24 hour followed by treatment with or without ASA at 5mM dose and cultured for 72 hour. We observed that PGF2α but not PGE2 significantly protected B16-MSCV cells from ASA-induced reduced survival  and left for 24 hour followed by treatment with 0.1% DMSO or 5mM ASA. After 72 hours, the analysis of SOX2 gene was performed by qRT-PCR and western blotting. As shown in Figure s Figure . Importas Figure  and apops Figure .We next investigated if SOX2 upregulation can rescue melanoma cells from ASA-induced inhibition of cell survival and induction of apoptosis. Notably, fibroblast growth factor-1 (FGF-1) treatment has been shown to upregulate SOX2 expression in murine primary calvarial osteoblast cells in a time dependent manner with substantial increase was noted at 24 hour . We firsAs ASA-induced effects were blocked by modulation of SOX2 by PGF2α and FGF-1 in melanoma cells Figures -7, we neAcetylsalicylic acid (ASA), the common non-steroidal anti-inflammatory (NSAID) household drug has been used for decades as an anti-inflammatory, analgesic and anti-pyretic agent , 50. In in-vitro and/or in-vivo melanoma growth [in-vitro and in-vivo growth of melanoma tumors via bypassing the PAF-R pathway. These studies are consistent with our published report demonstrating that augmentation of murine EL4 lymphoma tumor growth bypasses the PAF-R signaling [The present studies tested the role and mechanisms of ASA in highly aggressive and syngeneic B16F10 melanoma model. Notably, where B16F0 and B16F1 murine melanoma cells are non-aggressive, B16F10 cells are the highly aggressive murine melanoma model which resembles the malignant stage of advanced melanoma in humans. Importantly, published studies have used either B16F0 or B16F1 melanoma cells in determining ASA-induced effects on a growth –40. Our ignaling , 50, 52,ignaling –33, 35.Prostaglandin-endoperoxide synthase or COX is an enzyme with multiple isoforms (COX-1 and COX-2) that catalyze the conversion of arachidonic acid to prostaglandins (PGs), one of the major pathways implicated in mediating inflammatory responses and/or affecting tumor growth –26, 50. in-vitro and in-vivo growth of B16F10 melanoma, we took advantage of the PCR array analysis. Our studies demonstrate 10 fold downregulation of SOX2 gene by ASA treatment. Further qRT-PCR analysis of B16-MSCV tumors confirmed the PCR array data analysis for the expression of SOX2 gene.Notably, COX-dependent and independent pathways have been proposed for ASA-induced effects in various tumor types including B16F0 and B16F1 melanoma models , 43, 50.SOX2 gene encodes for a transcription factor which belong to SOX gene family and functions as an activator or suppressor of the gene transcription . Earlierin-vitro and in-vivo growth of highly aggressive B16F10 melanoma tumors via SOX2-dependent-PAF-R independent signaling pathway. Importantly, as SOX2 play crucial roles in tumor-resistance or anti-apoptotic responses to standard therapies, our studies indicate that anti-SOX2 targeting agent(s) could further be tested for melanoma chemoprevention.In summary, the present studies describe a novel mechanism by which ASA suppresses the 2 ProfilerTM PCR array were purchased from Qiagen Sciences . The cDNA kit and SYBR green qPCR reagent were purchased from Life technologies . The PAF-R, SOX2 and GAPDH primers were obtained from SABiosciences . Caspase-3/7 activity assay kit was purchased from Promega Corporation . Prostaglandin E2 (PGE2) and F2α (PGF2α) were purchased from Cayman Chemicals Co. . Antibodies against cleaved caspase-3, SOX2, β-actin were purchased from Cell Signaling Technology .All chemicals were obtained from Sigma-Aldrich  unless indicated otherwise. The Qiagen RNA extraction kit and RTμg/ml mixture of penicillin and streptomycin as described by us [Murine parent PAF-R negative B16F10 melanoma cells obtained from ATCC  used to generate stably PAF-R-expressing (B16-PAFR) and deficient (B16-MSCV) cells as described by us –18. Simied by us –19, 52 aPtafr−/−) mice  and PAF-R knockout mice (of Tokyo  on C57BL3 cells/well) and cultured overnight followed by treatment with 0.1% DMSO or various concentrations of ASA (1-10 mM) in serum-free media, as indicated in respective figures and figure legends. The cell survival was assessed at 24 hour, 48 hour and 72 hour by sulforhodamine-B (SRB) assay, as described by us previously [The B16-MSCV and B16-PAFR cells were seeded into 96-well plates (2×10eviously . In sepaeviously .4 cells/well) and treated with 10 nM 1-hexadecyl-2-N-methylcarbamoy l-3-glycerophosphocholine (CPAF) and/or 5mM ASA and incubated for 72 hour. The control cells received 0.1% DMSO. After 72 hour, cells were trypsinized, washed and resuspended in media. The cell proliferation was assessed by standard trypan blue exclusion method using a Countess automated cell counter  [The B16-MSCV and B16-PAFR cells were plated in 24-well plates  .5 cells/well) were seeded into 6-well plates with or without PGF2α (1.0 μg/ml) and after 24 hour treated with 0.1% DMSO or 5 mM ASA in serum-free media. After 72 hour, cells were homogenized in hypotonic extraction buffer and 0.5 mg of total protein was incubated with caspase-3/7 glo reagent  as described by us [The B16-MSCV or B16-PAF-R cells  were seeded in 100mm disc with or without PGF2α (1.0 μg/ml) and cultured for 24 hour followed by treatment with 0.1% DMSO (vehicle) or 5mM ASA in serum free media and left for 72 hours. Whole-cell extracts were prepared and cell lysates containing 40-60 μg proteins were subjected to SDS-PAGE and blotted onto PVDF membrane for western blot analysis as described by us previously [B16-PAF-R and/or B16-MSCV cells  to determine the dose of ASA that inhibits melanoma tumor growth. Mice were monitored for tumor development and mice weights were recorded. Finally, we choose 11.1 mmol/L (dissolved in water with 0.2% DMSO). Next, 0.5 × 106 murine B16-PAFR and B16-MSCV cells were implanted subcutaneously (SQ) into the shaved right hind flanks of WT and PAFR−/− mice (day 0) followed by treatment with or without ASA during the entire period of the experiment. Control mice received 0.2% DMSO in water as vehicle. Tumor growth (major and minor circumferences) in these studies was monitored and measured every 3 days with digital calipers , and tumor volume was calculated (major circumference × minor circumference2/2). Additionally, mice and tumor weights were recorded by digital balance.To determine effects of tumor versus stromal PAF-R expression in ASA-mediated modulation of experimental melanoma tumor growth, we first performed pilot studies with n=5 syngeneic WT mice and following 0.5 × 102 ProfilerTM PCR array plates (PAMM-176Zc-2 for mouse cancer stem cells) and qPCR and analysis were performed as per manual's protocol and software.To determine the ASA target(s), B16-MSCV tumors from vehicle and ASA-treated WT mice (n=10 mice/group) were harvested for PCR array analysis. Briefly, total RNA was extracted and 1μg of total RNA from each tumor sample was pooled separately from each group. Complementary DNA (cDNA) was synthesized using cDNA synthesis kit and blotted separately into RTin-vitro experiments were repeated independently at least three times and at least five mice/group were used in murine experiments. Data were analyzed by Student's t-test or one-way ANOVA with post hoc Bonferroni's or Newman-Keuls multiple comparison tests. Statistical significance was set as a p value <0.05.Statistical analysis was assessed by Graph Pad Prism 5.0 software. All


Kras-initiated lung and pancreatic tumours. In both models, p53 constrains progression to advanced adenocarcinomas. Comparison of lineage-related p53 knockout and wild-type clones reveals a minor role of p53 in suppressing cell expansion in lung adenomas. In contrast, p53 loss promotes both the initiation and expansion of low-grade pancreatic intraepithelial neoplasia (PanINs), likely through differential expression of the p53 regulator p19ARF. Strikingly, lineage-related cells are often dispersed in lung adenomas and PanINs, contrasting with more contiguous growth of advanced subclones. Together, these results support cancer type-specific suppressive roles of p53 in early tumour progression and offer insights into clonal growth patterns during tumour development.Although it has become increasingly clear that cancers display extensive cellular heterogeneity, the spatial growth dynamics of genetically distinct clones within developing solid tumours remain poorly understood. Here we leverage mosaic analysis with double markers (MADM) to trace subclonal populations retaining or lacking p53 within oncogenic  Using mosaic analysis with double markers to label genetically-distinct clones in established tumors, the authors studied the effects of p53 loss in lung and pancreatic cancers. They find that loss of p53 enhances progression in both models but only influences initiation in the pancreas. Cancer cells within developing tumours exhibit significant genetic and phenotypic heterogeneity mediating tumour growth, metastasis and therapy resistance124568Kras (KrasG12D) and biallelic inactivation of p53 in cells residing in the tissues of origin11LSL-KrasG12D/KrasWT; p53WT/WT and LSL-KrasG12D/KrasWT; p53flox/flox mice infected with inhaled adenovirus carrying Cre recombinase, our laboratory revealed a role of p53 in limiting tumour progression from low-grade lung adenomas to advanced adenocarcinomas14p53 mutation in regulating late-stage lung tumour progression. Finally, exome-sequencing analyses of murine lung adenocarcinomas derived from LSL-KrasG12D/KrasWT; p53flox/flox mice revealed no recurrent mutations beyond Kras and p53  or precursor PanINs of high-grade histology18p53 mutation shortens the latency and increases the frequency of PDAC formation in mouse pancreatic tumour models in which p53 is simultaneously mutated at the time of oncogenic Kras activation13Previous studies have also suggested that p53 principally plays a role late in pancreatic tumorigenesis. Similar to what is seen in human lung tumoursp53 loss of heterozygosity (LOH) following oncogenic Kras-mediated tumour initiation. We more faithfully model clonal evolution during tumorigenesis and perform high-resolution tracing of subclones lacking or retaining p53 during tumour progression. We demonstrate that sporadic p53 loss promotes progression to advanced lung and pancreatic tumours. Moreover, we confirm that p53 primarily plays a role late in lung tumorigenesis. In contrast, we determine that p53 suppresses both the initiation and expansion of early pancreatic tumours, which correlates with expression of the p53 regulator p19ARF. Finally, we show surprisingly significant intratumoral cell dispersion of subclones in early lung and pancreatic tumours.In this study, we adapt these models to permit sequential and sporadic p53 LOH in Kras-initiated tumors, we took advantage of mosaic analysis with double markers (MADM), which permits simultaneous fluorescence cell labelling and mutagenesis through a single Cre-mediated inter-chromosomal recombination event in mice22LSL-KrasG12D mice with MADM11-GT,p53WT/MADM11-TG,p53KO mice to generate LSL-KrasG12D/KrasWT; MADM11-GT,p53WT/MADM11-TG-p53KO mice (K-MADM-p53) (Methods). On Cre expression, oncogenic Kras is efficiently induced via intra-chromosomal Cre-mediated recombination permitting tumour initiation  of the MADM cassettes is predicted to result in the generation of two genotypically and phenotypically distinct daughter cells from a colourless p53KO/WTparent cell: GFP+/tdTomato− (green) p53KO/KO and GFP−/tdTomato+ (red) p53WT/WT  p53 LOH promotes lung tumour progression, we administered lentiviral Cre via the trachea to adult K-MADM-p53 mice to induce stable Cre expression in lung epithelial cellsp53KO/KO) cells did not predominate at early time points ERT2)  and displayed histologic features of adenocarcinoma . Together, these data are consistent with a role of p53 in constraining lung tumour progression to adenocarcinoma.We confirmed these findings using an alternative, less efficient MADM model in which el)ERT2) . This meanalysis . These tarcinoma . In addiWT cells , supportp53 also suppresses cell expansion in early lung tumours, we classified lung adenomas based on their green or red cell predominance in tissue sections of K-MADM-p53 mice at 10–16 weeks post infection (p.i.)  showed no colour dominance on qualitative analysis of random cross-sections of adenomas. Given the possibility for stochastic differences in individual daughter cell expansion following G2-X recombination, we did observe tumours (69 of 132) that showed green or red cell predominance. However, the proportions of green-dominant and red-dominant tumours were not statistically different, suggesting that green p53KO/KO cells did not have a selective growth advantage at this stage  . As green (p.i.) , green cis stage . To moreeks p.i. . These rp53 loss in K-MADM-p53 mice. To circumvent this limitation, we used the sum of all red and green single-labelled cells in a tumour as a surrogate for timing of G2-X recombination with the assumption that increased overall cell labelling indicates earlier time points of p53 LOH. If the duration of p53 loss altered cell expansion, we would expect a positive correlation between the total number of single-labelled cells and the green-to-red ratio. Interestingly, there was no association between these two parameters in lung adenomas  exhibited green, red and yellow acinar, ductal and islet cells but no overt cellular phenotypes due to p53 loss  mice harbouring homozygous p53 mutation (∼6 weeks) and heterozygous p53 mutation (∼16 weeks)   , support6 weeks) . We conf6 weeks) .p53 loss on tumour initiation in pancreatic cancer, we took advantage of the fact that G2-X recombination and labelling could occur during pancreatic development  containing predominantly green or red cells  before p53 mice . Interesp53 mice , suggested cells . Unlike ed cells , consistp53KO/KOcells exhibiting EdU incorporation was significantly increased compared with p53WT/WT cells in low-grade PanINs  and apoptosis (cleaved caspase-3) by immunostaining tissue sections of pancreatic and lung tumours. The percentage of e PanINs . In contgenotype . As low-ed cells . High-grporation , whereasporation . TogetheKras-driven lung and pancreatic cancer models harbouring a p53R172H mutant allele , which demonstrate similar histologic progression to the MADM models11p53 is stabilized, due in part to loss of feedback inhibition, and serves as a marker of endogenous p53 expressionWe hypothesized that differences in the timing of induction or stabilization of p53 protein expression may account for the functional differences observed between the tumour types. As wild-type p53 is difficult to detect by immunohistochemistry (IHC) on tissue sections with currently available antibodies, we took advantage of oncogenic Kras in tumour initiationLSL-KrasG12D; LSL-p53R172H mice, we observed expression of p19ARF in early- and late-stage pancreatic lesions in similar pattern to p53 expression , these invasive cells are likely to be derived from the PanIN epithelial cells. These findings are consistent with extratumoral invasion and epithelial-to-mesenchymal transition during putative preinvasive PanIN stages and corroborate previous work using Pdx1-Cre; LSL-KrasG12D/KrasWT; p53flox/WT mice harbouring a LSL-YFP reporterIn addition to pursuing quantitative analyses of tumour cell expansion, we also used the MADM models to monitor the spatial relationships between lineage-related cells during tumour progression. Using MADM, we observed green mesenchymal-like cells nearby but dispersed from green high-grade PanINs, some of which had lost expression of the epithelial marker cytokeratin 19 , in 6-weAside from the extratumoral spread of cells from putative preinvasive tumours in the pancreas, we also observed remarkable intratumoral dispersion of lineage-related cells in early lung and pancreatic tumours. Rather than exclusively displaying adjacent clusters of expanding green and red cells as would be expected with the stationary growth of epithelial tumour cells, lung adenomas and low-grade PanIN lesions often displayed subclones in which cells were non-contiguous in tissue sections . We confK-MADM-p53 mice at 10-16 weeks p.i. Although labelled cells of the same colour were separated by an average of ∼8.7 cell diameters , p53KO/KO cells surprisingly exhibited significantly decreased dispersal than p53WT/WT cells . Indeed, as p53KO/KO tumour cells progress, they appear to form more densely packed clones , MADM11-TG (Stock #013751), Pdx1-Cre (Stock #014647) and p53flox/flox (Stock #008462) mice were obtained from the Jackson Laboratory. MADM11-TG,p53WT/MADM11-TG,p53WT mice were crossed with p53KO/WT mice, which carry a p53-null allele lacking exons 2–6 , G12D/KrasWT; MADM11-GT,p53WT/MADM11-TG-p53KOPdx1-Cre; LSL-Kras (Pdx1-Cre-K-MADM-p53) and control mice lacking G12DLSL-Kras (Pdx1-Cre-MADM-p53) or p53 mutation (Pdx1-Cre-K-MADM) were generated by crossing the above breeders. LA2,Rosa26-CreERT2/KrasWT; MADM11-GT,p53WT/MADM11-TG-p53KO Kras and LA2,Rosa26-CreERT2/KrasWT; MADMKras (lacking p53 mutation) mice were generated in parallel crosses. G12D/KrasWTPdx1-Cre; LSL-Kras(KC); LSL-R172H/WTp53, KC; p53flox/WT, KC;LSL-R172H/floxp53 and flox/floxKC; p53 pancreatic cancer mice and G12D/KrasWTLSL-Kras; LSL-R172H/floxp53 and G12D/KrasWT; p53flox/floxLSL-Kras lung cancer mice were produced previously described1113pPGK-Cre lentiviral backbone and packaging vectors (delta8.2 and VSV-G) using TransIT LT-1 (Mirus Bio). Viral supernatant was collected at 48 and 72 h after transfection, filtered and concentrated by ultracentrifugation. Virus was resuspended in OptiMEM, titered and administered intratracheally to miceLentivirus was produced by co-transfection of 293T cells with KrasLA2,Rosa26-CreERT2/KrasWT; MADM-p53 and KrasLA2,Rosa26-CreERT2/KrasWT; MADM (lacking p53 mutation) mice at 5–10 weeks of age were injected intraperitoneally with tamoxifen (Sigma) at a dose of 9 mg per 40 g total body weight every other day for a total of three doses, to induce CreERT2-mediated recombination.−1 4,6-diamidino-2-phenylindole  for 1 h at room temperature (RT) or overnight at 4 °C and stored in cold PBS before imaging. For tissue section analyses, 5–30 μm sections were cut using a Leica cryostat, air dried for 30–60 min, washed three times with PBS, stained with DAPI (Life Technologies) for 5 min and mounted in Vectashield (Vector Labs) before imaging. For immunofluorescence staining, sections were blocked with 0.5% PNB (Perkin Elmer) and stained with primary antibody, donkey anti-rabbit Alexa 647 secondary antibody  and DAPI before mounting in Vectashield. Rabbit anti-CK19  and rabbit anti-cleaved caspase-3 (Asp175)  primary antibodies were used. EdU was detected in tissue sections using a Click-iT EdU Alexa Fluor 647 Imaging Kit (Thermo Fisher Scientific).MADM mice were sacrificed by CO2 asphyxiation and perfused with cold 4% paraformaldehyde  in PBS. For proliferation studies, mice were intraperitoneally injected with EdU (Setareh Biotech) 1.5 h before sacrifice. Tissues were dissected, fixed overnight with cold 4% PFA, cryoprotected with 30% sucrose and embedded in OCT (Tissue-Tek). For whole-mount multi-photon imaging, fixed tissues were liberated from OCT by washing with PBS, incubated with 1 μg ml°C. Samples were degassed for 10 min under nitrogen, incubated at 37 °C for 4 h, rotated in clearing solution (200 mM boric acid and 4% SDS pH 8.5) and subject to electrophoretic tissue clearing in a custom-built chamber for 48 h at 55 °C and 10–15 V. Samples were incubated in 0.1% Triton X-100 in PBS for 3 days followed by an additional 3 days in FocusClear (CelExplorer Labs) before imaging.CLARITY samples were prepared as previously describedp53R172H mutation exhibiting p53 protein accumulation due to loss of negative feedback.IHC for p53 and p19ARF was performed as previously describedPdx1-Cre-K-MADM-p53 mice were dissociated using a protease cocktail containing collagenase (Worthington), dispase (Roche) and trypsin-EDTA (Invitrogen) in Hank's balanced salt solution, quenched with fetal bovine serum (Hyclone) and passed through a 100 μm filter  before plating in DMEM (CellGro) containing 10% fetal bovine serum and penicillin/streptomycin (VWR). Cells were grown in culture for 2–3 days and subject to FACS analysis using a Guava flow cytometry system (Millipore). Stable GFP+/tdTomato− (green) cell lines were obtained through serial passage and confirmed to be p53KO/KO by PCR genotyping of genomic DNA isolated using QuickExtract (Epicentre). For immunocytochemistry, primary cultured cells were grown on glass coverslips, fixed with PFA, permeabilized with 0.2% Triton X-100, blocked with 5% BSA in PBS and stained with rabbit anti-p53 primary antibody , donkey anti-rabbit Alexa 647 secondary antibody  and DAPI before mounting onto glass slides with Vectashield. Unlike with tissue sections, endogenous p53 was readily detectable by immunofluorescence on cultured cells.Pancreatic tumour cells from Live imaging of cultured cells was performed with a Nikon Eclipse TE2000-U light microscope and SPOT RT3 camera. Immunocytochemistry and immunofluorescence tissue section images were obtained using an Andor camera attached to a Nikon 80 Eclipse 80i fluorescence microscope using × 4, × 10 and × 20 objectives. Additional immunofluorescence tissue section images were obtained with an Olympus FV1200 Laser Scanning Confocal Microscope with × 10, × 20 and × 30 objectives. Whole-mount images of intact fixed tissues were taken using a Nikon Eclipse TE2000-U dissecting light microscope and SPOT RT3 camera. CLARITY samples were imaged at a depth of up to 0.5 mm using a Nikon A1R Ultra-Fast Spectral Scanning Confocal Microscope. Multi-photon imaging was performed on an Olympus FV1000MP inverted microscope with a × 25, numerical aperture 1.05 objective and a SpectraPhysics Maitai tai/sapphire laser at 840 nm. Collagen 1, collected as second harmonic-generated polarized light, DAPI, green fluorescent protein (GFP) and tdTomato images were collected in three photomultiplier tubes (PMTs) with band-pass filters of 425/30, 425/45 and 607/70. Optical sections 5 μm apart to a tissue depth of 100–200 μm were obtained. IHC sections were imaged using a Nikon Digital Sight DS-U3 camera. Image processing, including merging colour channels, three-dimensional reconstructions and movie generation, was performed with ImageJ (NIH). n=132 total) from a single section from each of five lungs derived from K-MADM-p53 mice infected at 10-16 weeks p.i. with lentivirus were classified for colour dominance as green-dominant (n=38), red-dominant (n=31) or no dominance (n=51). An additional 12 tumours contained no labelled cells or only yellow cells without evidence of G2-X recombination. χ2-Test of green-dominant and red-dominant tumours was performed with the null hypothesis being an expected ratio of 1:1. All green and red cells from a random subset of tumours from 10 weeks p.i. (n=9 tumours) and 16 weeks p.i. (n=8 tumours) mice were quantified from alternating 30 μm sections through entire tumours . Green-to-red cell ratios were calculated for each tumour and geometric means with 95% confidence intervals were plotted. Total number of single-labelled cells (sum of green and red cells) versus green-to-red cell ratio was plotted for all tumours and R2 and P-values were calculated by linear regression.All lung adenomas (n=92) from a single section from each of five pancreata derived from 6-week-old Pdx1-Cre-K-MADM-p53 mice were classified for colour dominance as all green (n=19), green-dominant (n=47), no dominance (n=14), red-dominant (n=10) or all red (n=2)  . χ2-Testn=28) and lung adenocarcinomas (n=3) were imaged using a × 10 objective and the number of DAPI+ and EdU+ nuclei were quantified using ImageJ for a single image per lung adenoma and a total of eight images from lung adenocarcinomas (n=3 mice). The percentage of EdU+/DAPI+ cells from each image was calculated, averaged for each grade of tumour and compared by two-tailed Student's t-test. For low-grade PanINs, × 10 objective images were obtained (n=2 mice) and the number of total GFP+, EdU+/GFP+, total tdTomato+ and EdU+/tdTomato+ were manually counted. The percentage of EdU+/GFP+ and EdU+/tdTomato+ cells were calculated and compared by two-tailed Student's t-test.For quantification of EdU incorporation, lung adenomas  or Lepr , although these genotypes did not have an impact on the frequency of tumour types observed.Tumour spectrum analysis was performed on n=31) from a single tissue section from K-MADM-p53 mice infected at 10-16 weeks p.i. and normalized to the mean distance between adjacent cells on the same image. Green and red cell distances were compared by two-tailed paired Student's t-test. P<0.05 was used as level of significance for all statistical analyses.Cell-to-cell distance in lung adenomas was determined by calculating the mean distance between labelled nuclei based on Delauney triangulation using the Delauney Voronoi plugin for ImageJ. Two-dimensional mean distances were calculated separately for all green and red cells within tumours .Supplementary Figures 1-3 and Supplementary Tables 1-2Movie of 3D reconstruction of multi-photon images taken of lung adenoma from Figure 4e showing dispersed green and red cells.Movie of 3D reconstruction of multi-photon images of additional lung adenoma with dispersed cells.Movie of 3D reconstruction of multi-photon images taken of lowgrade PanIN from Figure 4f showing dispersed green and red cells.Movie of 3D reconstruction of multi-photon images of additional low-grade PanIN with dispersed cells. Note the presence of colorless and yellow cells interspersed between green cells in this PanIN.


RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.RIT1 belongs to the RAS family of small GTPases. Germline and somatic  RIT1 have been identified in NS. RIT1 belongs to the RAS superfamily, however, the cellular function of RIT1 remains elusive. We show that RIT1 binds p21-activated kinase 1 (PAK1), an effector of the RHO GTPases RAC1 and CDC42, which are important regulators of cytoskeletal dynamics. NS-associated RIT1 mutants enhance complex formation between RIT1, RAC1/CDC42 and PAK1. Expression of wild-type or mutant forms of RIT1 caused loss of stress fibers and mature focal adhesions and enhanced cell motility. Our data suggest that dysfunction in actin dynamics is a novel aspect in the pathophysiology of RASopathies.Noonan syndrome (NS) belongs to the RASopathies, a group of developmental diseases caused by mutations in genes encoding RAS-MAPK pathway components. Germline mutations in  RIT1 belongs to the RAS superfamily of low molecular weight GTP-binding proteins that function as guanine nucleotide-regulated molecular switches in the cell by changing between an active GTP-bound and an inactive GDP-bound state . The RASRIT1 mutations in patients with Noonan syndrome (NS) demonstrated the importance of this small GTPase for embryonic development , GST-PLCE1[RA] and GST-PIK3CA[RBD] (as positive control) under all tested cell culture conditions . As a neRit1 has been found in a protein complex with Rac/Cdc42 and Par6 , 45. p21RIT1 mutations affect formation of the novel RIT1-PAK1 complex. We pulled down HA-tagged RIT1 wildtype or mutants from HEK293T lysates by using PAK[CRIB]. Compared with wild-type RIT1, RIT1K23N and RIT1G95A significantly increased the amount of co-precipitated RIT1  and from cells cultured in 10% serum   . Taken tThe major function of RAC1, CDC42, and PAK1 is the regulation of the actin cytoskeleton to modulate cell shape, motility and adhesion –50. ActiIt has been reported that loss of stress fibers promoted by Pak1 was accompanied by disappearance of focal adhesions , 52, andK299A, RAC1S17N and CDC42S17N mutants on actin stress fiber turnover and found no effect on stress fiber distribution and morphology in COS7 cells (Expression of constitutively active forms of RAC1 and CDC42 (Q61L) also was associated with loss of stress fibers, and parallel blocking of PAK1’s activity prevented stress fiber dissolution , 58. We S7 cells . Co-exprS7 cells , and expS7 cells . These dRIT1 construct  of PI3K (PIK3CA) (amino acids 127–314), the RAS-association (RA) domain of RALGDS (amino acids 777–872), the RA domain of PLC1 (PLCE1) (amino acids 2130–2240) and the CRIB domain of PAK1 (amino acids 58–141) were used to specifically pull down GTP-bound RIT1 from cell extracts. Preparation of GST-RBD/RA/CRIB beads, cell lysis and precipitation of GTP-bound GTPases have been described previously .Transiently transfected HEK293T cells were cultured as indicated in the respective experiment. Cells were lysed in ice-cold co-immunoprecipitation buffer , and cell lysates were clarified by centrifugation. RAC1 immunoprecipitation shown in E. coli strain BL21 transformed with pGEX-2TK-PAK[CRIB], pGEX-4T3-CDC42 and pGEX-4T3-RAC1, respectively, as described previously , GDP/GTPγS-loaded GST-CDC42 and GDP/GTPγS-loaded GST-RAC1 fusion proteins coupled to glutathione-bound agarose beads were mixed with different concentrations of GDP/GTPγS-loaded His-RIT1WT/A57G/F82L protein—as indicated in the respective experiment—in binding buffer . After incubation for 30 min at room temperature, precipitates were collected by centrifugation and washed with binding buffer (4 times) and proteins were analyzed by western blotting. Purified GST-SAPAP2 (NM_004745.4) (expressed from plasmid pGEX-6P-1) was a gift from Stefan Kindler .GST-PAK[CRIB], GST-CDC42 (amino acids 1–178) and GST-RAC1 (amino acids 1–181) fusion proteins were isolated from http://rsb.info.nih.gov/ij/index.html).Coverslips were coated with 10 μg/ml collagen type I  in PBS for 1 hour at room temperature. Excess collagen was removed, COS7 cells were seeded on coverslips, transfected with expression constructs and serum-deprived overnight. Subsequently, cells were rinsed with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS and washed three times with PBS. After treatment with permeabilization/blocking solution , cells were incubated in antibody solution (3% goat serum and 0.1% Nonidet P40 in PBS) containing appropriate primary antibodies. Cells were washed with PBS and incubated with Fluorophore-conjugated secondary antibodies or Texas Red-X phalloidin (Life Technologies) in antibody solution. After extensive washing with PBS cells were embedded in ProLong Diamond Antifade Mountant with DAPI  on microscopic slides. Cells were examined in epifluorescence mode of an Olympus cell tool TIRFM system equipped with a 60x oil immersion objective lens and pictures were taken of representative cells to visualize the observed morphological changes. Phalloidin-stained cells were categorized in two different groups: cells with normal and cells with reduced or absent actin stress fibers (exemplary photographs are shown in 2 growth factor reduced Matrigel Basement Membrane Matrix (Corning) according to manufacturer´s instructions. Briefly, 105 HEK293T cells transiently expressing various RIT1 protein variants were seeded in transwell upper compartment in a volume of 100 μl of serum-free DMEM. For chemo attraction, 600 μl of DMEM supplemented with 10% FBS were added into the lower compartment. 48 h later cells that moved to the lower compartment were carefully detached by trypsinization and transferred to FACS tubes. Cell numbers were determined by flow cytometry using AccuCheck Counting Beads (Thermo Fisher Scientific).Cell invasion was investigated by using Costar Transwell cell culture inserts for 24 well plates with 8 μm pores (Corning), which were coated with 100 μg/cmTransfection efficiency was determined by staining of cells with anti-HA-fluorescein antibody and quantification of HA-fluorescein positive cells. To determine cell numbers, detached cells were washed twice with PBS. To each FACS tube, 20 ml of AccuCheck Counting Beads (Thermo Fisher Scientific) was added. Flow cytometry was carried out using a FACS Calibur (BD Biosciences). In each sample, 3000 beads were acquired and the number of cells, which were collected in the meanwhile, was determined using CellQuestPro Software (BD Biosciences).http://rsb.info.nih.gov/ij/index.html). Quantitative data are presented as the mean ± standard deviation (SD) performed by GraphPad prism7 software . Statistical significance was assessed by Student's t-test for pairwise comparisons or one-way ANOVA for multiple comparisons. In the latter case, statistically significant differences were identified by post hoc analysis using Student's t-test followed by Bonferroni correction. Data shown in graphs of P value <0.05.Signals on autoradiographs were quantified by densitometric analysis using the ImageJ software Click here for additional data file.S1 Figt-tests were used to determine statistical significance . (C) HEK293T cells were transfected with empty vector (EV) or HA-tagged RIT1 expression constructs (wildtype [WT] and p.G31R) as indicated and cultured under steady-state condition (10% serum). Total cell lysates were analyzed as described in (A). Two independent experiments (Exp. #1 and #2) are shown.(A and B) HEK293T cells were transfected with empty vector (EV) and constructs expressing HA-RIT1 wildtype (WT), HA-RIT1 p.K23N, p.G31R, p.F82L, p.M90V or p.G95A as indicated. Cells were cultured under serum-starved condition  and serum-starved condition followed by 5, 15, or 30 min stimulation with 20% serum. Total cell lysates were analyzed by immunoblotting using anti-phospho-ERK1/2 (pERK1/2) (A and B) and anti-ERK1/2 (ERK1/2) antibodies (A). Expression of RIT1 protein variants was monitored by immunoblotting using anti-HA antibody, and anti-GAPDH antibody was applied to control for equal loading (A). Data shown are representative of three independent experiments. (B) Immunoblots from three independent experiments (Exp.) demonstrate that the RIT1 p.G95A mutant stimulates ERK1/2 phosphorylation under serum-starved condition (0 min). The immunoblot shown in Exp. 1 is the same as the one in (TIF)Click here for additional data file.S2 FigSer473 (pAKTSer473) and anti-AKT (AKT) antibodies. Expression of RIT1 protein variants was monitored by immunoblotting using anti-HA antibody, and anti-GAPDH antibody was used to control for equal loading. Data shown are representative of three independent experiments. (B) HEK293T cells were transfected with empty vector (EV) or a construct expressing HA-RIT1 wildtype (WT), cultured under serum-starved condition  and serum-starved condition followed by 5, 15, or 30 min stimulation with 10 ng/ml EGF. Total cell lysates were analyzed by immunoblotting using anti-phospho-AKTThr308 (pAKTThr308) and anti-AKT (AKT) antibodies. Expression of HA-tagged RIT1 protein was monitored by immunoblotting using anti-HA antibody, and anti-GAPDH antibody was used to control for equal loading. Data shown are representative of three independent experiments.(A) HEK293T cells were transfected with empty vector (EV) and constructs expressing HA-RIT1 wildtype (WT), HA-RIT1 p.A57G, p.F82L or p.G95A as indicated. Cells were cultured under serum-starved condition  and serum-starved condition followed by 5, 15, or 30 min stimulation with 20% serum. Total cell lysates were analyzed by immunoblotting using anti-phospho-AKT(TIF)Click here for additional data file.S3 Figt-tests were used to determine statistical significance. ns, not significant. (B) One-way ANOVA between groups: P < 0.05; ns, not significant.(A and B) HEK293T cells were transfected with empty vector (EV) and RIT1 expression constructs as indicated and cultured under serum deprivation  or basal condition . Endogenous PAK1 was precipitated with an anti-PAK1 antibody . As IP control, an irrelevant isotype-matched antibody (anti-pSMAD2 antibody) was used (IP: ctrl). Enrichment of PAK1 in the precipitates and the amount of endogenous PAK1 in TCL was demonstrated with an anti-PAK1 antibody (#2). Co-precipitated HA-RIT1 and expression of HA-RIT1 in total cell lysates (TCL) was detected by an anti-HA antibody. (C) COS7 cells were transfected with empty vector (EV), RIT1 wildtype or p.G95A expression constructs as indicated and cultured under serum deprivation (0.1% serum). Endogenous PAK1 was precipitated with an anti-PAK1 antibody [IP: PAK1 (#1)]. Enrichment of PAK1 in the precipitates and the amount of endogenous PAK1 in TCL was demonstrated with an anti-PAK1 antibody (#1). Co-precipitated HA-RIT1 and expression of HA-RIT1 in TCL was detected by immunoblotting using an anti-HA antibody. Juxtaposed autoradiographs derive from the same western blotting membrane. (D) HEK293T cells expressing GFP-PAK4 and either HA-tagged RIT1 WT or the HA-RIT1 p.G95A mutant were cultured under serum deprivation (0.1% serum). GFP-PAK4 was precipitated by GFP trap. Enrichment of GFP-PAK4 (GFP trap) is shown by direct comparison of precipitates and TCL. HA-RIT1 was detected by using an anti-HA antibody. As positive IP control, co-precipitated endogenous CDC42 was detected by an anti-CDC42 antibody. Data are representative for three independent experiments.(TIF)Click here for additional data file.S5 FigP < 0.05; ns, not significant.(A and B) HEK293T cells were transfected with empty vector (EV) and RIT1 expression constructs as indicated and cultured under basal condition (10% serum). Endogenous CDC42 and RAC1 were immunoprecipitated with an anti-CDC42 antibody [CDC42 (#3)] (A) and an anti-RAC1 antibody (B), respectively. Co-precipitated HA-RIT1 and HA-RIT in total cell lysates (TCL) was detected by an anti-HA antibody. Enrichment of CDC42 and RAC1 in the precipitates and amount of endogenous CDC42 and RAC1 in TCL was demonstrated with an anti-CDC42 antibody [CDC42 (#4)] (A) and an anti-RAC1/2/3 antibody (B), respectively. Data shown for CDC42 and RAC1 are representative of three (A) and four (B) independent experiments, respectively. Autoradiographic signals were quantified by scanning densitometry. The amount of co-precipitated HA-RIT1 was double-normalized relative to amounts of immunoprecipitated CDC42/RAC1 and HA-RIT1 in total cell lysates. To conserve the relative variance of the samples, values for RIT1 wildtype and RIT1 mutants were divided by the mean of the wildtype samples . The gra(TIF)Click here for additional data file.S6 FigG31R or RIT1A57G was immoprecipitated with an anti-CDC42 antibody [IP: CDC42 (#5)]. Endogenous CDC42 protein in total cell lysates (TCL) and precipitates was detected with an anti-CDC42 antibody [WB: CDC42 (#4)]. Co-precipitated HA-RIT1 and HA-RIT1 in TCL was detected by an anti-HA antibody. As a control, an isotype-matched non-specific anti-IgG rabbit antibody (IgG) was used for immunoprecipitation. The asterisk indicates the light chain of the antibody used for precipitation.(A and B) HEK293T cells were co-transfected with MYC-empty vector (MYC-EV), MYC-CDC42 (A) or MYC-RAC1 (B) expression construct together with HA-empty vector (HA-EV), RIT1 wildtype (WT) or RIT1 p.G95A expression construct and cultured under serum deprivation (0.1% serum). Lysates were split for precipitation using either anti-MYC coupled beads (Trap MYC in A and B) or primary antibodies against CDC42 (IP CDC42 in A) and RAC1 (IP RAC1 in B). As IP control for primary antibodies an irrelevant isotype-matched antibody against V5 (V5) was used. Both in precipitates  and total cell lysates (TCL), RIT1 protein was monitored by immunoblotting using an anti-HA antibody, RAC1 and CDC42 were detected by an anti-MYC antibody. Anti-GAPDH antibody was used to control for equal loading. Results are representative for two independent experiments. (C) HEK293T cells were co-transfected with EGFP-empty vector (EGFP-EV) or an EGFP-RHOA expression construct (EGFP-RHOA) together with HA-empty vector (HA-EV), RIT1 wildtype (WT) or RIT1 p.G95A expression construct and cultured under serum deprivation (0.1% serum). Enrichment of EGFP or EGFP-RHOA by EGFP trap (Trap EGFP) is shown by direct comparison of precipitates and total cell lysates (TCL). HA-RIT1 was detected by using an anti-HA antibody and EGFP and EGFP-RHOA by an anti-EGFP antibody. Asterisk indicates the light chain of the antibody used for precipitation. Data are representative for three independent experiments. (D) Endogenous CDC42 of stably transfected Flp-In 293 cells expressing RIT1(TIF)Click here for additional data file.S7 FigRecombinant His-tagged RIT1 wildtype (0.5 μM) was loaded with GDP or non-hydrolyzable GTPγS as indicated and incubated with 1 μM GST-CDC42 (loaded with GDP or GTPγS) (A) or GST-RAC1 (loaded with GDP or GTPγS) (B). Recombinant GST (1 μM) and GST-PAK[CRIB] (1 μM) were used as negative and positive control, respectively. Glutathione agarose-coupled GST fusion proteins were precipitated and samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). Data shown are representative of three independent experiments.(TIF)Click here for additional data file.S8 FigWT construct and serum-starved overnight. Cells were stained with mouse anti-HA-antibody followed by anti-mouse Alexa Fluor 488-conjugated antibody, Texas Red-X Phalloidin for actin distribution and DAPI for nucleus staining. White boxes indicate magnified parts of specimen shown on the right-hand side. Scale bars, 10 μm. Exemplary images depicting cells with normal, reduced or absent actin stress fibers are shown.(A) COS7 and HEK293T cells were plated on collagen-coated glass slides and kept under serum starvation overnight. F-actin was visualized using Texas Red-X Phalloidin. Paxillin was stained using mouse anti-paxillin antibody and Alexa Fluor488-conjugated anti-mouse antibody. Nuclear DNA was labeled by DAPI. White boxes indicate magnified parts of specimen shown on the very right-hand side. Scale bar, 10 μm. (B) COS7 cells were plated onto collagen-coated coverslips, transiently transfected with the HA-RIT(TIF)Click here for additional data file.S9 Fig(A) COS7 cells were plated on collagen-coated glass slides, transiently transfected with the indicated construct and kept under serum starvation overnight. HA-tagged RIT1 was stained by rabbit anti-HA antibody followed by anti-rabbit Alexa Fluor546-conjugated antibody. Paxillin was visualized using mouse anti-paxillin antibody and Alexa Fluor488-conjugated anti-mouse antibody. Nuclear DNA was labeled by DAPI. Scale bar, 10 μm. (B) The number of nascent (dot-like) and mature (stripe-like) paxillin-positive structures per cell was determined in at least 30 cells of each dataset.(TIF)Click here for additional data file.S10 FigS17N (A) and RAC1S17N (B) were stained by mouse anti-MYC antibody followed by anti-mouse Alexa Fluor488-conjugated antibody. HA-tagged RIT1 was visualized using rabbit anti-HA antibody and anti-rabbit Alexa Fluor647-conjugated antibody. F-actin was stained by using Texas Red-X Phalloidin, and nuclear DNA was labeled by DAPI. The label “HA” indicates cell(s) not transfected with the RIT1 wildtype construct. For co-expression of RAC1S17N and RIT1 wildtype two separate microscopic data series are shown (last two rows in B). White boxes indicate magnified parts of specimen shown on the very right-hand side. Scale bar, 10 μm.COS7 cells were plated on collagen-coated glass slides, transiently transfected with the indicated constructs and serum-starved overnight. MYC-tagged CDC42(TIF)Click here for additional data file.S11 FigHEK293T cells were transiently transfected with cDNA constructs expressing the indicated HA-tagged RIT1 protein variants and serum-starved overnight. Cells were stained with anti-HA-fluorescein antibody and counted by flow cytometry. Percentage of cells positive for HA-fluorescein staining is indicated for each construct.(TIF)Click here for additional data file.


Fetuin-A is a biomarker of disease activity in multiple sclerosis. Our aim was to investigate whether Fetuin-A plays a direct role in the neuroinflammatory response in the mouse EAE model. Peak Fetuin-A expression in the CNS and in peripheral lymphoid tissue correlated with peak EAE disease activity. Fetuin-A-deficient mice showed reduced EAE severity associated with an accumulation of splenic monocyte and dendritic cell populations, increased IL-12p40, ASC1, and IL-1β expression, and an increase in T regulatory cells. The upregulation of Fetuin-A in LPS-stimulated dendritic cells and microglia further supports an intrinsic role of Fetuin-A in regulating innate immune activation during EAE. Multiple sclerosis (MS) is a chronic autoimmune disease with an uncertain etiology characterized in the majority of patients by recurring episodes of immune-mediated demyelination in the CNS. Chronic demyelination over time leads to an accumulation of axonal loss, glial scarring, grey matter pathology, and progressive neurological disability affecting approximately 3 million people worldwide. A major objective at the time of initial diagnosis is to minimize future disability by arresting the disease early and effectively, thereby preventing later disease progression and disability. With many immune-targeting disease modifying treatments available, the challenge in assessing therapeutic intervention is the accuracy of the clinical parameters used to monitor disease activity, namely relapse rate, disability score, and MRI. There is a need for biomarkers that can detect disease activity that lies below the threshold of our current measurement tools.Fetuin-A was recently described as a novel biomarker of disease activity in the cerebrospinal fluid (CSF) of patients with MS . CSF levFetuin-A, a glycoprotein named based its abundance in fetal serum , is exprThe mechanism by which Fetuin-A may influence EAE or MS pathogenesis remains unknown. Fetuin-A is a multifunctional protein shown to have both anti-inflammatory and pro-inflammatory effects . Fetuin-All animal experiments were approved by the St. Luke’s Roosevelt Hospital Center IACUC. Heterozygous fetuin-A knockout mice have been described previously . Wild-ty® Array Mouse Immune Response 96-well Plate (Life Technologies) and fold changes in gene expression were determined by RT2 Profiler PCR Array Data Analysis software (Qiagen).For gene expression analysis, mice were perfused with PBS and spleens and spinal cords removed and stored in RNAlater (Qiagen). For time course experiments, spinal cords were cut longitudinally and half of the tissue stored in RNAlater. Total RNA was isolated using RNeasy Lipid Tissue Mini Kit  or RNeasy Mini Kit  (Qiagen) and reverse transcribed using Superscript VILO Master Mix (Life Technologies). Quantitative real time PCR (qRT-PCR) was performed using TaqMan Universal PCR Mater Mix with prevalidated TaqMan gene expression assays (Life Technologies) to detect ASC (Pycard) (Mm00445747_g1), CCL2 (Mm00441242_m1), CCL5 (Mm01302427_m1), CCL7 (Mm00443113_m1), CCL19 (Mm00839967_g1), CCR4 (Mm01963217_u1), CCR7 (Mm01301785_m1), FoxP3 (Mm00475164_m1), Fetuin-A (Mm00442674_m1), Gata3 (Mm00484683_m1), H2Eb1 (Mm00439221_m1), ICAM1 (Mm00516023_m1), IFNγ (Mm00801778_m1), IL-1β (Mm00434228_m1), IL-10 (Mm00439614_m1), IL-12a (Mm00434169_m1), IL-12b (Mm00434174_m1), IL-17 (Mm00439618_m1), IL-23a (Mm00518984_m1), LTα (Mm00440228_gH), MBP (Mm01266402_m1), NFκB (Mm00476361_m1), RORc (Mm01261022_m1), Tbx21 (Mm00450960_m1), TGFβ (Mm00441724_m1), TLR4 (Mm00445273_m1), TNFα (Mm00443260_m1). Hprt1 (Mm00446968_m1) was used as an endogenous control. PCR was performed using 7900HT Fast Real-Time PCR System and results analyzed using RQ Manager software (Life Technologies). Lymph node qPCR was performed using TaqMan® Bis-Tris polyacrylamide gels (Life Technologies) and Western blot performed with 50 ng/ml goat anti-mouse Fetuin-A antibody (R&D Systems) or anti-β-actin (Sigma), followed by HRP conjugated secondary antibody. Bands were visualized by chemiluminescence, and bands were quantitated by densitometry and normalized to β-actin. Band intensities for each time point were normalized to day 0 which was set to a value of 1.For time course analysis of protein expression, the other half of the spinal cord tissue was snap frozen. Tissue was lysed in RIPA buffer and protein concentration determined by BCA protein assay (Pierce). Proteins were separated using NuPAGEEAE mice were perfused with 4% paraformaldehyde on days post-immunization (DPI) as indicated in the legend. Brain and spinal cord were removed and paraffin embedded. Transverse spinal cord sections were analyzed by immunofluorescence using anti-mouse-Fetuin-A (R&D Systems), anti-CD3 (Abcam), anti-MBP (Covance), anti-Iba-1 (WAKO), followed by Alexa-488, Alexa-555, or Alexa-647-conjugated secondary antibodies (Life Technologies). Nuclei were visualized using 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence imaging was performed by confocal microscopy (Zeiss LSM 510).Quantitation of lesion number and immune cell infiltration was performed by a blinded observer on eight individual transverse cross-sections of spinal cord per animal spanning the length of the spinal cord. Lesions were identified as areas of demyelination (reduced MBP staining) in each individual spinal cord section and counted. For CD3 and Iba-1 quantification, the number of cells positive for each marker were counted manually for all lesions and expressed relative to the tissue area.+/CD25+/FoxP3+ cells. Analysis was performed on a FACSAria flow cytometer (BD Biosciences).Mice were perfused with PBS and spinal cords were removed and dissociated using Neural Tissue Dissociation Kit (Miltenyi). Single cells were washed and centrifuged at 300 x g for 10 minutes. For myelin removal, cells were resuspended in cold 0.9 M sucrose in HBSS and centrifuged at 850 x g for 10 minutes. Supernatant containing myelin was removed by aspiration, and cells were washed in PBS and centrifuged for 3 minutes at 850 x g. To harvest spleens, mice were sacrificed by cervical dislocation, and spleens removed, dissociated using gentleMACS dissociator (Miltenyi) and strained through a 40 μm cell strainer. Red blood cells were lysed using ACK buffer (Life Technologies) and cells washed twice with PBS. Cells were resuspended in 2%FBS in PBS and Fc block (BD Pharmingen) and stained with CD45-PE-Cy7 (30 F11), CD4-FITC (RM4-5), CD8-APC (53–6.7), CD14-FITC (Sa2-8), CD11b-APC (M1/70), CD11c-APC (N418), MHC-II-PE-Cy7 (M5/114.15.2), CD80-FITC (16-10A1) , and CD19-PE-Cy7 (1D3), Ly6C-FITC (AL-21)  antibodies as described in figure legends. FITC, APC and PE-Cy7-conjugated isotypes were used as controls for gating. Mouse Regulatory T cell staining kit (ebiosciences) was used to analyze CD4+ dendritic cells were isolated from mouse bone marrow and cultured as described previously [+ by flow cytometry. CD11b+ primary microglia cells were isolated from brain tissue of 5-day old pups. Brain tissue was dissociated using Neural Tissue Dissociation Kit (Miltenyi) and CD11b+ cells were positively selected using magnetic cell sorting (Miltenyi). One day after plating, cells were stimulated with LPS as above for 4 or 48 hours.Spleens from WT EAE mice were dissociated and CD11c+ and CD11b+ cells were isolated sequentially by positive selection using magnetic cell sorting following manufacturer’s instructions (Miltenyi). CD11ct-test. Statistical significance was set to p values <0.05. GraphPad Prism 5 was used to calculate significance.Differences between daily EAE scores, average area under the curve (AUC), and daily weights in WT and FAKO groups were analyzed by Mann-Whitney test. Data with multiple groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post test. Data with two groups were analyzed using unpaired, two-tailed, Student’s Our previous study suggested a role for Fetuin-A during EAE onset and severity, which was associated with Fetuin-A immunostaining in degenerating neurons around demyelinated spinal cord lesions in mice during late stage EAE (day 32 post-immunization) . To betthi/CD11b-) and macrophage (CD45hi/CD11b+) infiltration compared to WT EAE mice (lo/CD11b+) was not significantly different between the two groups of mice.To further evaluate the contribution of Fetuin-A in EAE development, we utilized mice lacking Fetuin-A , which we previously reported had delayed EAE onset, reduced disease severity, and reduced degree of spinal cord demyelination examined on day 32 . BecauseEAE mice . The perImmunofluorescent staining of EAE spinal cords also showed reduced CD3 (T cell) and Iba-1 (macrophage/microglia) immunoreactivity that appeared to be confined to the meninges and perivascular spaces  comparedThe immunological gene expression profile in the CNS of WT and FAKO EAE mice revealed that genes associated with innate immune response and antigen presentation, such as TGFβ, LTα, TNFα, NFκB, TLR4, and H2-Eb1 were significantly reduced in FAKO EAE compared to WT EAE . Genes aThe expression of Fetuin-A in both the spleen and CNS during classical EAE suggests that Fetuin-A may play a dual role in peripheral immune response and in ongoing neuroinflammation. To address the contribution of Fetuin-A to peripheral immune activation in EAE, we utilized the Th17-driven adoptive transfer model of EAE whereby splenocytes from EAE mice (WT or FAKO) are restimulated with MOG peptide, IL-12, and anti-IFN-γ antibody in order to expand Th17 cells while preventing Th1 . Injecti+/CD25+/FoxP3+ regulatory T cells  . Validat+ and CD11b+ cells. As shown in + and CD11c+ cells (data not shown). In contrast, Fetuin-A was not detectable in CD11b-/CD11c- flow-through cells, or in naïve, Th1-polarized, Th17-polarized, or in MOG-restimulated T-cells (data not shown). To further explore the expression of Fetuin-A in CD11c+ cells, we cultured CD11c+ bone marrow dendritic cells (BMDCs) from either WT or FAKO mice. In WT-BMDCs, Fetuin-A mRNA was detectable and significantly upregulated approximately 3.5-fold upon stimulation with LPS  Naïve CD4+ T cells from Fetuin-A-deficient mouse splenocytes were capable of Th17 polarization when compared to WT cells. Control cells were stimulated with anti-CD3/CD28 and IL-2 only, and Th17 polarized cells were stimulated with anti-CD3/CD28, IL-2, IL-1β, IL-23, TGF-β, IL-6, anti-mouse IL-4, and anti-mouse IFN-γ. Percentage of CD4+/IL-17+ positive cells was determined by flow cytometry. (B) There was no difference in proliferation in WT or FAKO-derived T cells when stimulated under control or Th17-polarizing conditions. Added Fetuin-A had no effect on proliferation. Proliferation was measured by reduced mean fluorescence intensity (MFI) of CFSE-labeled cells when compared to unstimulated controls.  Addition of Fetuin-A does not affect Th17 cell polarization of human T cells when analyzed by expression of Th17-associated transcription factor RORc (C) or IL-17 cytokine production (D).  Addition of Fetuin-A does not affect Treg cell polarization of human T cells when analyzed by expression of Treg-associated transcription factor FoxP3 (E) or percentage of CD4+/FoxP3+ T cells (F).(TIF)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.