Cell lines wereprofiled using array comparative genomic hybridisation, Illumina gene expressionanalysis, reverse phase protein arrays, and targeted sequencing ofKRAS hotspot mutations. Frequent gains were observed at 2p, 3q,5p, 7p, 7q, 8q, 12p, 13q, 14q, and 17q and losses at 2q, 3p, 5q, 8p, 9p, 9q, 14q,18q, and 20p. Frequently gained regions contained EGFR,PIK3CA, MYC, SMO,TRIB1, FZD1, and BRCA2, whilefrequently lost regions contained FHIT and MACROD2.TRIB1 was selected for further study. Gene enrichment analysisshowed that differentially expressed genes with respect to treatment response wereinvolved in Wnt signalling, EGF receptor signalling, apoptosis, cell cycle, andangiogenesis. Stepwise integration of copy number and gene expression data yielded 47candidate genes that were significantly correlated. PDCD6 wasdifferentially expressed in all three treatment responses. Tissue microarrays wereconstructed for a cohort of 118 CRC patients and TRIB1 andMYC amplifications were measured using fluorescence insitu hybridisation. TRIB1 and MYC wereamplified in 14.5% and 7.4% of the cohort, respectively, and these amplificationswere significantly correlated (p≤0.0001). TRIB1 proteinexpression in the patient cohort was significantly correlated with pERK, Akt, andCaspase 3 expression. In conclusion, a set of candidate predictive biomarkers for5-fluorouracil, oxaliplatin, and BEZ235 are described that warrant further study.Amplification of the putative oncogene TRIB1 has been described forthe first time in a cohort of CRC patients.Selecting colorectal cancer (CRC) patients likely to respond to therapy remains aclinical challenge. The objectives of this study were to establish which genes weredifferentially expressed with respect to treatment sensitivity and relate this tocopy number in a panel of 15 CRC cell lines. Copy number variations of the identifiedgenes were assessed in a cohort of CRCs. IC Colorectal cancer (CRC) accounts for 8% of all cancer deaths , with vaAPC (70–80%),TP53 (50%), KRAS (35–45%),PIK3CA (25–32%), BRAF (10–17%) andPTEN (4–5%) . Th. Th® nitescribed .™ RPPA AnalysisModule software (VigeneTech Inc.).The RPPA spots were quantified using MicroVigene®DNA Variant Analysis Software V3.97 . Theraw data files .ab1 generated by the ABI Prism® 3100 GeneticAnalyzer  were imported into the software and thedefault analysis settings were applied. The GenBank annotation files wereautomatically downloaded and the reference files used for mutation detection wereautomatically synthesised.Sanger sequencing data were analysed using Mutation Surveyor® GenomicSuite™ Version 6.6 (Partek Inc.). The data were initiallynormalised using Loess Normalization and the Genomic Segmentation algorithm wasused to analyse the copy number amplifications and deletions. The customsegmentation parameters were as follows: the minimum genomic markers was 10, thep-value was 0.001, and the signal-to-noise ratio was 0.03. A region was reportedas lost if the log2 copy number ratio was below -0.3 and gained if the log 2 copynumber ratio was above 0.15. Three different region lists were created: (1)regions that were gained in seven or more cell lines; (2) regions deleted in sevenor more cell lines; (3) those containing the highest amplifications, i.e., log2ratio equal or greater to 1.0 . Additionally,genomic segmentation clustering was performed using Euclidean distance and averagelinkage. The copy number analysis was conducted on chromosome 1 to chromosome 22and excluded the two sex chromosomes.aCGH data were analysed using PartekThe sample gene profile file generated from the gene expression analysis wasquantile normalised and filtered for those probes where the detection p-value≤0.05. The data were then log2 transformed and mean centred to obtainrelative values between the cell lines. The sample gene profile file was thenannotated using Hg18 prior to performing differential gene expression analysis(DGEA).vs. 5-FU less sensitive cell lines, where highly sensitivecell lines were defined as having an IC50 ≤ 30μM; (2)L-OHP highly sensitive cell lines vs. L-OHP less sensitive celllines, where highly sensitive cell lines were defined as having an IC50≤ 10μM; (3) BEZ235 sensitive cell lines vs. BEZ235insensitive cell lines, where sensitive cell lines were defined as having anIC50 < 80nM.The DGEA was performed using ArrayMining, an online microarray data miningsoftware package .DiffereInterpretation of data was accomplished using Functional Annotation Clustering inDAVID bioinformatics resources .The gene expression data for the genes located in the frequently gained regionswas filtered out. Using Pearson’s correlation coefficients with Bonferronicorrection, a list of genes that had a significant correlation between the log2copy number value and gene expression was generated. The gene expression data forcell lines were analysed with respect to treatment response using Mann-Whitney Utest using GraphPad Prism 6.Data generated from RPPA were normalised using Cluster 3.0, an open sourceclustering tool . DataweFive-micron haematoxylin and eosin-stained slides were prepared from the FFPE blocks,and tumor areas were marked by a pathologist and a trained research technician.Following histopathological examination, 118 cases were chosen out of the originalcohort and a tissue microarray (TMA) was constructed by a qualified technician. Fourbiological replicates (TMA000034A-D) were constructed as described in detailelsewhere  and cut Protein expression of TRIB1 was assessed with anti-TRIB1 rabbit polyclonal antibodyin the CRC TMA using Automated QUantitative Analysis (AQUA), described in detailelsewhere ,29. TRIBTRIB1 and MYC amplification in the CRC patientcohort were investigated using fluorescence in situ hybridisation(FISH). A MYC/CEN8p probe was purchased from Abnova (cat. no. FG0065) and theTRIB1/CEN8p probe was custom designed by Abnova. The protease treatment time wasvaried to optimise digestion and ensure good quality hybridisation. Visualisation wasperformed using DAPI  to stainnuclei.MYC and TRIB1were purchased from Abnova. The MYC/CEN8p FISH probe consisted of an ~160kbMYC probe located at 8q24.12-q24.13 with a Texas Red fluorophoretogether with an ~520kb CEN8p probe located at 8p11.21 with a FITC fluorophore. TheTRIB1/CEN8p FISH probe consisted of an ~260kb TRIB1 probe located at8q24.13 with a Texas Red fluorophore together with an ~520kb CEN8p probe located at8p11.21 with a FITC fluorophore.Ready-to-use dual-labelled probes for Scoring was carried out by a trained technician and a consultant pathologist. Theslides were scored using a Leica DMLB fluorescent microscope using 100X oil immersionlens. The Colorado Scoring Criteria were used  to scorehttp://www.tmanavigator.org/) was used for statistical analysis.Survival analysis for TRIB1 and MYC amplificationin the CRC cohort was carried out using GraphPad Prism 6.TRIB1 protein expression data generated from AQUA analysis were correlated with AKT,caspase 3, cyclin B1, ERK, Ki67, MYC, S6, PTEN, pAKT, pERK, pHistone H3, pMEK, andpS6 protein expression. Statistical analysis was carried out using Pearson’scorrelations, and p-values were adjusted for multiple testing using the Bonferronicorrection. An open source programme TMA Navigator  for 96 h, thirteen CRC cell lines showedvarying degrees of sensitivity when treated with drug concentrations ranging from2.5μM to 100μM . Two CRCeficient . This preficient .in vitro studies have suggested thatTP53 deficiency contributes to drug resistance [TP53 deficient (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), but theywere still sensitive to 5-FU in this study. Mariadason et al., however, reported nodifference in 5-FU-induced apoptosis in mutant and wild type p53 cell lines [Although a number of sistance , we failll lines .50 values for L-OHP ranged from 3.0 to 31.1μM,with a median of 8.0 μM, demonstrating a ten-fold range of sensitivity. Themost sensitive cell lines were HT29, LS411N, and SW837, while the least sensitivewere Colo320DM, NCI H716, and Colo201. No statistical significance  was observed when comparing L-OHP IC50 values between dMMRcell lines and pMMR cell lines, which is in agreement with a similar study by Fink etal. [After treatment with oxaliplatin (L-OHP) for 96 h, the CRC cell lines showed varyingdegrees of sensitivity when treated with increasing concentrations of L-OHP rangingfrom 2.5μM to 100μM . The IC5k etal. .50 values , in contrast to a previous report [TP53mutational status was not associated with benefit from first-line oxaliplatin-basedtreatment [There was no association between p53 status and L-OHP ICs report . Howeverreatment .50 valuesfor BEZ235 ranged from 13.4 to >80nM, with the sensitive cell lines having amedian sensitive concentration of 23.6nM. The most sensitive cell lines were HT29,Colo201, and NCI H716, while NCI H508, T84, SW48, SW480, Colo320DM, HCT116, HCT116p53-/-, and LoVo were insensitive at a concentration of 80nM. No statisticallysignificant difference was observed  between theIC50 values for BEZ235 treatment and PIK3CA mutant andwild type groups. All the PI3KCA mutant cell lines had either aBRAF or a KRAS co-mutation. No COSMIC data wasavailable for MTOR mutations in these cell lines (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/). Serra etal. established that BEZ235 arrested proliferation in all 21 cancer cell lines usedin their study, independent of PI3K pathway mutation status [BRAF or KRAS mutation or EGFRamplification were slightly less sensitive to BEZ235 compared to the other cell lines[Seven CRC cell lines were sensitive and eight CRC cell lines were insensitive totreatment with various concentrations (2.5nM and 80nM) of BEZ235 for 96 h . The IC5n status , and thall lines.KRAS exon 2mutations. The DLD-1, HCT116, HCT116 p53-/-, and LoVo cell lines had a 5574G>A substitution consistent with a G13D missense mutation; the SK-CO-1 andSW480 cell lines had a 5571 G>T substitution consistent with a G12V missensemutation; SW837 had a 5570 G>T substitution consistent with a G12C mutation;and T84 had a 5574 G>A substitution consistent with a G13D mutation. This isin agreement with published sequencing data and data in the COSMIC database(http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/). There wereno statistically significant differences in response to 5-FU, L-OHP, and BEZ235 withrespect to KRAS mutational status .Of the 15 CRC cell lines, eight cell lines possessed 50 values for 5-FU, L-OHP,and BEZ235 using Pearson’s correlations with complete linkage showed that thecell lines did not cluster according to any particular mutation. There wasvariability in response to the three different treatments , focaladhesion ,apoptosis , and regulation of transcription . Important KEGG pathwaysassociated with 5-FU mode of action and subsequently enriched in the list originatingfrom this study included purine metabolism , pyrimidine metabolism , drug metabolism (GSTO2), ABCtransporters (ABCB1), and oxidative phosphorylation(NDUFA9).Genes differentially expressed with respect to 5-FU sensitivity are listed in GLI2, GLI4, SETDB2,NFXL1, POLE4, PURA,TSNAX, ZBTB41, ZNF20,ZNF254, ZNF420, ZNF689,ZNF7, ZNF91), regulation of transcription, regulation of cell cycle, and apoptosis  (ATP6V1B2), Jak-STAT signalling pathway(CBLC), hedgehog signalling pathway (GLI2),glycolysis (AKR1A1), glutathione metabolism(GSTO2), drug metabolism (GSTO2), cysteine andmethionine metabolism (MTAP), MAPK signalling pathway, base excision repair, andnucleotide excision repair (POLE4) pathways were enriched in thisgene set.Genes differentially expressed with respect to L-OHP sensitivity were involved withDNA binding ( TNFSF9) . OxidatiCPS1, G6PD,PYGL), cell death , responseto drug ,chromatin organization , regulation of transcription , and DNA binding  , phosphatidylinositol signalling system(PIK3C2B), and the Jak-STAT signalling pathway(SPRY1) were enriched in less sensitive cell lines.The most differentially expressed genes with respect to BEZ235 sensitivity wereinvolved in glucose metabolism (ZNF75A) . The WntA total of 971 genes were located in frequently gained regions, of whichcorresponding gene expression data were available for 667 genes. A total of 47 geneswere significantly correlated and are listed in TBRG4 (p ≤ 0.001),MRPL32 (p ≤ 0.001), CYCS (p ≤0.001), PDCD6 (p = 0.01), COBL (p = 0.01),DDX56 (p = 0.01), MRPS17 (p = 0.01),PDS5B (p = 0.03), TOMM7 (p = 0.03),AEBP2 (p = 0.04), NOD1 (p = 0.04),MIR1204 (p = 0.04) and RFC3 (p = 0.05).Significant differences were found between response to BEZ235 treatment and geneexpression of PDCD6 (p = 0.002), MYC (p = 0.01),MRPL32 (p = 0.01), TBRG4 (p = 0.03) andPURB (p = 0.04). Significant differences were found betweenresponse to L-OHP treatment and gene expression of PDS5B (p <0.005), UBL3 (p = 0.01), MTIF3 (p = 0.02),CASC8 (p = 0.02), XPO4 (p = 0.04),GTF3A (p = 0.04) and PDCD6 (p = 0.04).20/47 of these genes were associated with treatment responses Figs  and 6. SReverse phase protein array (RPPA) was used to measure protein expression of 31phosphorylated and non-phosphorylated proteins in the CRC cell lines. Two mainsub-clusters were produced by unsupervised hierarchical clustering of RPPA data. Sub-cluSignificant differences in protein expression were found for FAK (p = 0.004) andphospho MEK (p = 0.005) with respect to 5-FU treatment responses . SignifiTRIB1, which was also observed to berecurrently amplified and overexpressed in a CRC study carried out by Camps et al.[Statistically significant correlations between copy number gains and gene expressionwere identified on amplicons located on chromosome 8. Candidate genes that could beinvestigated further included s et al.. Furthers et al.. TRIB1 hs et al.. This res et al.. TRIB1 is et al., 49. Furs et al., one of s et al.,transfos et al., apoptoss et al.. In views et al..TRIB1region  and mRNA expression ofTRIB1 . TheTRIB1 region was gained in seven cell lines and clearly amplifiedand very highly expressed in NCI H716 cells. Reverse phase protein array (RPPA)analysis of TRIB1 was carried for the cell lines, which did not reveal a correlationwith log2 copy number ratio  or with gene expression. Nevertheless, a large variation between TRIB1protein expression was observed between the different cell lines that did not reachstatistical significance.There was a weak correlation between DNA copy number of the (R) [TRIB1 copy numberin a cohort of 881 CRC patients , where TRIB1 wasfound to be gained in 11% of primary CRC samples. Consequently, the amplification ofTRIB1 and MYC in the tissue microarrayconsisting of 118 Dukes’ A and B CRC patients was analysed.The Oncomine(R)  databaseTRIB1. FISH scores forTRIB1 ranged between 0.45 and 3.38 . Of 76 cases, 11 tumors (14.4%) were amplified (a score of≥1.8).Of the 118 cores (each representing a case), a total of 76 cores contained nucleithat could be scored for MYC. FISH scores for MYC ranged between 0.70 and4.14 . Six tumors were amplified forMYC (7.4%).Of 118 cores, a total of 81 cores contained nuclei that could be scored forTRIB1 and MYC FISH scores were strongly positivelycorrelated .TRIB1 protein expression was next investigated using AQUA. Protein expression in thecytoplasm and nucleus was successfully measured in 96 out of 118 cases. Five samplesout of the 96 samples showed TRIB1 overexpression (5.2%) in the cytoplasm whenconsidering a cut-off of two standard deviations, while 6/96 showed TRIB1overexpression (6.25%) in the nucleus.TRIB1 or MYCamplifications and those without.Of 22 proteins in the MAPK pathway, TRIB1 protein expression in the cytoplasm wassignificantly correlated (p = 0.05) with TRIB1 (nucleus), phospho-Erk, Akt, Myc(nucleus), PTEN (cytoplasm), cleaved caspase 3 (nucleus), and phospho-MEK (nucleus),after correcting for multiple testing. TRIB1 protein expression in the nucleus wassignificantly correlated (p = 0.05) with TRIB1 (cytoplasm), Akt, phospho-Erk, and Myc(nucleus), after correcting for multiple testing . There wAlthough the mutation status of a number of individual candidate genes has beenassociated with responses to CRC therapy, the results are inconclusive and few haveresulted in useful stratification biomarkers. Here, the cellular response to treatmentwith 5-FU, L-OHP and BEZ235 was not associated with the mutational status of commongenes in multiple cell lines. The measurement of a mutation in a single gene alone wasinsufficient to stratify patients for CRC therapy, which argues for adopting amulti-scale approach to help identify other factors that contribute to therapeuticresistance.BCL2, DCC, CTDP1,SMAD2, and FHIT [BCL2 is not usually considered to be a tumorsuppressor gene, it has been reported to act as one under certain circumstances [MACROD2 at 20p12.1 which was alsodescribed in a recently published study by Linnebacher et al. [The list of tumor suppressor genes found in this study’s frequently deletedregions included and FHIT –60. Althmstances . Furtherr et al. .Systematic analysis of copy number gains allowed us to identify regions that were gainedin seven or more cell lines. The use of a high-resolution array allowed analysis offrequently amplified regions that contained less well described genes. This analysis,when combined with gene and protein expression analysis and extensive literature review,helped us to identify a number of genes that could be further investigated as possiblenovel oncogenic drivers and determinants of response to therapy.PDCD6)gene expression than less sensitive cell lines. PDCD6, located oncytoband 5p15.33-p14.1, is known to be involved in apoptosis survival [PDCD6 accumulates in the nucleus andinduces apoptosis in response to DNA damage [PDCD6 inhibits angiogenesis through the PI3K/mToR/p70S6K pathway byinteracting with VEGFR-2 [1 phase of cell cycle progression and apoptosis involvedin human ovarian tumorigenesis. This study suggested that suppressingPDCD6 supports tumorigenesis by inhibiting apoptosis in ovariancancer [A number of genes were amplified, overexpressed, and associated with therapeuticresponses. By adopting a functional multiscale analytical approach, a list of 20candidate predictive biomarkers for 5-FU, L-OHP, and BEZ235 was generated.5-FU-sensitive cell lines had higher programmed cell death 6  loss elicited chemotherapeuticresistance in lung cancer [Seven proteins were associated with responses to cytotoxic therapies, but nodifferential expression was seen with the PI3K/mTOR inhibitor. For example, focaladhesion kinase (FAK) was differentially expressed between 5-FU and L-OHP groups. FAK isassociated with apoptosis and proliferation pathways in cancer cell lines . Cdc2 wag cancer ,while cg cancer .TRIB1, was selected for further analysis as acandidate biomarker. There was a highly statistically significant correlation betweenthe FISH score of TRIB1 and MYC , consistent with co-amplification. A number of studies have suggestedthat MYC-driven cancers are reliant on other genes and pathways, unlikenon-MYC-driven cancers [TRIB1. Furthermore, TRIB1 appearsto be druggable, involved in oncogenic pathways, and differential toxicity. Geneexpression silencing of TRIB1 using deconvoluted siRNA pool-mediatedknockdown resulted in increase in cleaved caspase 3 and 7 and in increase ofγ-H2AX foci in c-MYC expressing human foreskin fibroblasts but not in the controlfibroblasts [As proof of concept of adopting a functional multi-scale analytical approach tocomprehend the underlying changes driving colorectal carcinogenesis, a gene that wasfrequently amplified,  cancers –90. Toyoroblasts .MYC and TRIB1 are co-amplified in anumber of CRC patients and that targeting TRIB1 would lead to celldeath via a synthetic lethal mechanism. Since MYC cannot betherapeutically targeted, it would be useful to investigate the function ofTRIB1 and its role in targeted therapy. MYC isknown to interact with a number of signalling pathways and is mostly involved in growthand proliferation. Furthermore, although MYC is prominently referred toas a proto-oncogene, MYC also exhibits pro-apoptotic properties [TRIB1 is targeted, MYC might function asa tumor suppressor gene leading to cell death. This would need to be validated in aseries of functional experiments.We speculate that operties . It is fIn addition, TRIB1 protein expression was significantly correlated with MYC,phosphorylated MEK, ERK, total Akt, PTEN, and cleaved caspase 3, consistent withprevious findings that TRIB1 interacts with MEK1 and overexpression leads to ERKphosphorylation .FurtherAlthough a difference in TRIB1 expression was observed in the cell line panel, there wasno statistically significant correlation between gene and protein expression. This couldhave occurred for a number of technical, statistical, and biological reasons includingassay sensitivity, array probe specificity, mRNA and protein degradation , and samThis study has a number of limitations. Further data analysis needs to be performed withrespect to frequently deleted regions to identify putative tumor suppressor genesinvolved in CRC and their relationship with treatment responses. This study usedcontinuous cancer cell lines, which may not fully represent parent tumors and,therefore, clinical responses to therapy. Nevertheless, cell lines have been shown torecapitulate the molecular and phenotypic characteristics of primary tumors –101, incTRIB1 is co-amplified withMYC in a proportion of CRCs and may be an attractive target forintervention in this group of patients.Our multi-scale analytical approach has generated a list of 20 candidate predictivebiomarkers for 5-FU, L-OHP, and BEZ235. This approach is valuable for understanding themode of action of different treatments and guiding personalised therapy. Furthermore, weshow, for the first time, that S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


The replication of virus is dependent on the host metabolism; nucleotide precursors for the synthesis of viral genome or viral mRNAs are supplied from the host nucleotides pool. As viral replication requires a massive synthesis of viral mRNA or viral genome, the inhibition of cellular nucleotide synthesis is considered a strategy for broad-spectrum antivirals. Indeed, the inhibition of GTP synthesis showed antiviral effects against many viruses. Mycophenolic acid (MPA) has broad-spectrum antiviral activity and the mechanism is to decrease cellular GTP concentration by inhibiting inosine-5’-monophosphate dehydrogenase (IMPDH), which catalyzes the rate-limiting reaction of de novo GTP biosynthesis . Many IMOn the other hand, pyrimidine synthesis inhibitors are getting more attention as a novel antiviral strategy. Several pyrimidine synthesis inhibitors have been discovered as active hit compounds from high-throughput screenings for antivirals –8. UnlikDespite this prominent antiviral effect in vitro, none of the pyrimidine synthesis inhibitors have shown antiviral effect in vivo models using mice –8. For tIn this study, we sought to understand better why pyrimidine synthesis inhibitors are not successful in inhibiting virus replication in mouse models. During the study of a novel pyrimidine inhibitor as a broad-spectrum antiviral, we observed results that are similar to Marianne et al. in that cells treated with pyrimidine synthesis inhibitors reduced virus replication significantly. More interestingly, we found that such antiviral effect was cell line-specific: i.e., human cell lines established an antiviral state by the treatment of pyrimidine synthesis inhibitors, but mouse cell lines did not. This observation could explain the lack of antiviral effect of pyrimidine synthesis inhibitors in mouse models. This finding may imply the fundamental difference in the mechanism of innate immune system in response to the inhibition of pyrimidine biosynthesis between human and mouse.50 for each. Monensin inhibits the acidification of endosome, which is required for the viruses to infect and release the genetic materials into the cytoplasm [To test whether pyrimidine synthesis inhibitors can inhibit virus replication in mouse cells in vitro, we measured the antiviral activity of brequinar and monensin in several cell lines through determining an ECytoplasm . Brequinytoplasm . The celytoplasm . pVSV-luytoplasm .The antiviral activities of brequinar and monensin in various cell lines are summarized in 50s were ~1 µM for both viruses, which is around 30-fold higher than those in 293T cells. Interestingly, brequinar did not show a strong antiviral activity in SY-SH5S cell line with an EC50 of 1.13 µM, even though it is a human cell line. These data clearly indicate that antiviral activity of brequinar is much less efficient in mouse cells and is cell line-dependent. These responses were the same with other stains of VEEV, such as V3526 or TC-83 (data not shown).In contrast to monensin, the antiviral activities of brequinar were cell line-specific. The EC50s of brequinar in 293T cells (human) were 0.031~0.039 µM for the viruses, implying a strong antiviral activity against both viruses in these cell lines. The antiviral activities in murine cell lines we tested , however, were much less compared to in 293T cell lines. The ECTo test whether the cell type-dependent antiviral activity of brequinar is associated with the induction of ISGs, we measured the induction of Interferon-Induced Protein With Tetratricopeptide Repeats 1 (IFIT1) gene as a marker for ISGs in two cell lines; 293T (responsive to brequinar) and Neuro 2A (less responsive to brequinar) after a treatment with brequinar for 18 hours . TreatmeThe antiviral activity associated with pyrimidine synthesis inhibition is expected to depend on two mechanisms: 1) direct effect from decreased pyrimidine concentrations for viral RNA and DNA synthesis, and 2) indirect effect through the induction of ISGs. It is difficult to measure the sole impact of each mechanism on the viral replication, as the latter mechanism is dependent on the first mechanism. The silencing of IRF1, which induces the ISGs, abrogated antiviral effect of brequinar; hence, the second mechanism seems more important for the antiviral activity .The lower antiviral activity of brequinar that was observed in mouse cells could be due to the binding specificity to mouse DHODH. All mammalian DHODHs are very similar and clustered in Family 2 group. The sequence identity between mouse and human DHODH is 88% . HoweverAn alternative hypothesis could be an absence of a pathway to activate ISGs in response to decreased cellular pyrimidine concentration. While both SH-SY5S and 293T cell lines are human cell lines, the antiviral activity of brequinar in the two cell lines were clearly different (~30-fold difference). Experiments with other pyrimidine synthesis inhibitors showed a lack of antiviral activity in the mouse cell lines (data not shown). The absence of an antiviral effect in mice regardless of their structure suggests a difference downstream pathway is involved. Currently, the pathway is unknown and we are currently focused on identifying of the nature of the pathway in both human and mouse.Aside from the mechanism, this study sheds light on selecting a proper animal model to evaluate antiviral activity of pyrimidine synthesis inhibitors. Many in vivo antiviral efficacy models use mice as the first line model for the convenience and virus susceptibility –20. In f


BRAF mutational status. Analysis of different signaling pathways affected by sorafenib may contribute to assist a more specific therapy choice with fewer side effects. Twelve thyroid carcinoma cell lines derived from anaplastic, follicular and papillary thyroid carcinomas with wildtype or mutationally activated BRAF were treated with sorafenib. Growth inhibition, cell cycle arrest, cell death induction and inhibition of intracellular signaling pathways were then comprehensively analyzed.Patients with dedifferentiated or anaplastic thyroid carcinomas currently lack appropriate treatment options. Kinase inhibitors are among the most promising new agents as alternative strategies. The BRAF- and multi-kinase inhibitor, sorafenib, has already shown antitumor effects in thyroid carcinoma patients in a phase III clinical trial. In this study we aim to better characterize molecular effects and efficacy of sorafenib against thyroid carcinoma cells with various histological origins and different Cell viability was analyzed by MTT assay, and the cell cycle was assessed by flow cytometry after propidium iodide staining. Cell death was assessed by lactate dehydrogenase liberation assays, caspase activity assays and subG1 peak determinations. Inhibition of intracellular pathways was analyzed in dot blot and western blot analyses.BRAFV600E allele were slightly more sensitive to sorafenib than those harboring wildtype BRAF. Cell cycle analyses and caspase assays showed a sorafenib-dependent induction of apoptosis in all cell lines, whereas increased lactate dehydrogenase release suggested cell membrane disruption. Sorafenib treatment caused a rapid inhibition of various MAP kinases in addition to inhibiting AKT and receptor tyrosine kinases.Sorafenib inhibited proliferation of all thyroid carcinoma cell lines tested with IC50 values ranging between 1.85 and 4.2 μM. Cells derived from papillary carcinoma harboring the mutant BRAF status, confirming that sorafenib is therapeutically beneficial for patients with any subtype of dedifferentiated thyroid cancer. Inhibition of single intracellular targets of sorafenib in thyroid carcinoma cells may allow the development of more specific therapeutic intervention with less side effects.Sorafenib inhibited multiple intracellular signaling pathways in thyroid carcinoma cells, which resulted in cell cycle arrest and the initiation of apoptosis. Sorafenib was effective against all thyroid carcinoma cell lines regardless of their tumor subtype origin or  Thyroid carcinoma originating from thyroid follicular cell is the most common endocrine malignancy ,2. AboutBRAF gene (mostly BRAFV600E), an effector of MEK that in turn activates the ERK1 and ERK2 mitogen-activated protein kinases  kinase signaling cascade (RAS-RAF-mitogen-activated protein kinase kinase (MEK)- extracellular-signal regulated kinase (ERK)) frequently occur in thyroid carcinomas ,3. About Review ,10). BRA. BRABRAF of ATCs , but in  of ATCs . The BRAcurrence . Mutatio .Inappropriate activation of the MEK-ERK kinase cascade leads to deregulated cell proliferation, dedifferentiation and improved cell survival in a variety of tumor cell types . The impin vitro . Moreovein vitro . Sorafenin vitro ,23,24. Smutation . These emutation . Furthermutation  showed tmutation . Taken tSome clinical phase II trials and clinical studies in patients with metastatic differentiated thyroid carcinomas have shown promising results for sorafenib -32. The BRAFV600E mutation), follicular or anaplastic subtypes. The aim of the present study was to elucidate the effects of sorafenib treatment on proliferation, cell death induction and intracellular signaling pathways in various thyroid carcinoma cell lines.Since sorafenib as a multikinase inhibitor blocks various intracellular signaling pathways, significant side effects have also been reported in clinical trials . A broadSorafenib  was provided by Bayer Health Care , stored in 10 mM aliquots in DMSO at −20°C and further diluted in the appropriate medium. Antibodies to detect both total protein and activated phosphorylated forms of c-Jun N-terminal kinase (JNK), AKT, p44/42 MAP kinase (ERK1/2) and p38 MAPK were purchased from Cell Signaling Technology .2.Cell lines derived from the anaplastic, papillary and follicular thyroid cancer subtypes were used in this study. The SW1736 , HTh7 3, HTh74 , HTh83 , and C64BRAF gene were described elsewhere [2, 1.5U HotMaster Taq polymerase  and 300 nM each of forward and reverse primers. Cycling conditions were 40 cycles of 94°C for 20 sec, 55°C for 10 sec, 65°C for 35 sec. PCR products were analyzed on 3% agarose gels and purified using the QIA quick removal kit (Qiagen). Sequencing was performed using the ABI Prism BigDye Terminator Cycle sequencing kit v1.1 on an ABI Prism 3100 Genetic Analyzer . Sequences were compared to the wildtype sequences using the Sequencher software .Genomic DNA was isolated from cell lines using the QIAamp DNA kit  according to the manufacturer’s instructions. Primers used to amplify exon 15 of the lsewhere . For PCR4 to 5 × 104 cells (cell line dependent) were seeded into 96-well plates containing the appropriate growth medium. Medium was replaced after 24 hours with culture medium without FBS but containing 0.1% bovine serum albumin (BSA) and the indicated sorafenib concentrations was added. After 48 hours, viable cells were stained with the Cell Titer Aqueous One Solution assay , and optical density at 490 nm was measured using an Emax microplate photometer . Control values without sorafenib treatment were performed as 22-fold determinations, while all concentrations of sorafenib were tested in 8-fold. Calculation of results and Student’s t-test were performed using SoftMax pro software (Molecular Devices), and IC50 values were calculated using Sigma Plot software .For proliferation assays, 1 × 104 to 5 × 104 cells (cell line dependent) were seeded into black, transparent-bottomed 96-well plates containing the appropriate growth medium. Medium was removed after 24 h and 100 μl culture medium without FBS, but containing 0.1% BSA and the denoted sorafenib concentration, was added to each well. After 14 or 24 hours, 50 μl of medium from each well was transferred to a fresh black 96-well plate and equilibrated to 20°C. According to the manufacturer’s instructions, 50 μl of CytoTox reagent was added and reactions were incubated for 10 min in the dark. After adding 25 μl of stop solution, fluorescence was determined with excitation and emission wavelengths of 560 nm and 590 nm, respectively. Wells containing no cells, as the zero setting, and fully lysed cells, as the maximum LDH release control, were included in each experiment. Caspase 3 and 7 activity in treated cells was determined in the original stimulation plate by adding 50 μl of Apo-ONE reagent that contained a fluorometric substrate in cell lysis and reagent buffer. After 60 min, fluorescence was measured at 521 nm after excitation with 499 nm. All values were performed as 8-fold determinations. Calculation of results and Student’s t-tests were performed using SoftMax pro software (Molecular Devices).Release of lactate dehydrogenase (LDH) from cells with damaged membranes was measured by the CytoTox-ONE homogeneous membrane integrity assay (Promega). Activity of caspases 3 and 7 was measured by the Apo-ONE homogeneous Caspase 3/7 assay (Promega). 1 × 105 to 5 × 105 cells/well in 6-well plates in appropriate growth medium for cell cycle analyses. Medium was replaced with medium without FBS but containing 0.1% BSA and 3 μM sorafenib 24 h later and cells were treated for the indicated times. Treated cells were harvested and fixed in cold 70% ethanol. RNase A (60 μg/ml) and propidium iodide (25 μg/ml) in PBS were added, and samples were incubated 20 minutes in the dark at room temperature. Samples were measured on a FACS Calibur flow cytometer , and cell cycle stages were analyzed using the ModFit Software .Cells were plated at 1 × 10Proteome Profiler™ antibody arrays  and western blotting were used to assess inhibitory effects of sorafenib on intracellular signaling proteins and receptor tyrosine kinases. Cells were plated in 10 cm culture dishes, and grown for 1–2 days to 85 to 90% confluency. Medium was removed and cells were washed once and maintained in prewarmed HBSS buffer (Life Technologies) for 20 minutes before adding 3 μM sorafenib. Treated cells were washed with ice-cold PBS, and all further steps were performed on ice. Cells were lysed in lysis buffer containing cOmplete protease inhibitor and phosSTOP phosphatase inhibitor cocktails . Lysates were clarified by centrifugation at 10,000 × g for 10 min at 4°C, protein concentration determined by modified Bradford assay  and 500 μg of protein from each lysate were used in dot blot analysis according to the manufacturer’s instructions. For western blotting, 30 μg of total protein was denatured by boiling for 5 minutes in SDS sample buffer, then separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories). After blocking with 5% skim milk powder or 5% BSA in TBS, blots were incubated with the appropriate primary antibody in TBS buffer containing 0.1% Triton X-100 (TBS-T) overnight at 4°C. After washing, an appropriate secondary antibody coupled to horseradish peroxidase in TBS-T was added. Bound antigens on western and dot blots were detected using the ECL Advance chemiluminescence detection kit . Signal intensity was evaluated with a CCD camera system, and differences were calculated with the Quantity One software (Bio-Rad Laboratories).Statistical analysis of treatment versus control groups was performed by means of the unpaired Student’s t-test using SPSS  or the other software packages indicated above. P-values < 0.05 were considered statistically significant.BRAFV600E mutational status for 48 h with a range of sorafenib concentrations or vehicle and assessed proliferative activity. We first assessed the mutational status of exon 15 of the BRAF gene in all cell lines using PCR. The BHT101 and B-CPAP papillary cell lines both harbored a heterozygous BRAFV600E mutation. The anaplastic cell line, SW1736 also harbored a heterozygous BRAFV600E mutation which is suggestive that the anaplastic tumor has originated from a papillary carcinoma , while SW1736 cells (anaplastic cell line with BRAFV600E mutation) had a midrange IC50 of 3.25 μM. TPC1 cells, which are derived from PTC but harbor no BRAFV600E mutation, had an IC50 value in the lower range that was slightly higher than those of the other 2 papillary cell lines, which harbor BRAFV600E mutations. The follicular cell lines, FTC133, FTC236, ML1 and TT2609, and the C643 and HTh7 ATC cell lines also had midrange IC50 values . HTh83 ATC cells and FTC238 FTC cells were most insensitive to sorafenib, with IC50s of 3.95 and 4.2 μM, respectively. While no dramatic differences were observed in the sensitivity of cell lines from different histological origins or with or without the BRAFV600E activating mutations to sorafenib, some trends were observed. The three papillary cell lines had the lowest overall IC50 values, and the two papillary cell lines harboring the BRAFV600E mutation (BHT101 and B-CPAP) were slightly more sensitive than TPC1 cells. However, the only BRAFV600E mutation harboring anaplastic cell line (SW1736) had an IC50 in midrange of the IC50s for all 4 anaplastic cell lines. These results indicate that BRAF activation does not play any role in undifferentiated carcinoma cells. Cell lines from follicular carcinomas, with exception of the relatively insensitive FTC238 cells, had midrange IC50 values suggesting that sorafenib targets kinases other than BRAF in these cells. Results for one representative cell line of all histological origins with or without BRAFV600E mutation (SW1736 cells (anaplastic with BRAFV600E mutation), HTh7 (anaplastic without BRAFV600E mutation), BHT101 (papillary with BRAFV600E mutation) and ML1 (follicular without BRAFV600E mutation) are depicted in Figure Sorafenib treatment decreased the number of viable cells in all 12 thyroid carcinoma cell lines analyzed, and efficiency of sorafenib did not span a large range, since IC50 values for all 12 cell lines were between 1.85 μM and 4.2 μM (Table BRAFV600E mutation) cells  and ML1 follicular cells were the most insensitive cell lines with respect to the lowest effective concentration of sorafenib  and in the SW1736 and HTh7 anaplastic cell lines , VEGFR2 (KDR) and PDGFRB in all four cell lines Table . PhosphoBRAFV600E mutations. The effects of sorafenib on various intracellular signaling molecules were studied to evaluate more specific treatment options in patients with dedifferentiated thyroid carcinomas patients. We assessed the BRAFV600E mutational status for all 12 cell lines used in this study. The activating BRAFV600E mutation was only detected in two of the three papillary cell lines (BHT101 and B-CPAP) and in one of the four cell lines (SW1736) as previously detected and reported [BRAF. These findings fit well with experimental and pathological evidence indicating an involvement of BRAF mutation in the pathogenesis of about 50% of PTCs and the progression of PTC to ATC, but no occurrence of BRAF mutations in FTC [Here we present a detailed analysis of kinase inhibition, effects on the cell cycle and apoptosis induction by the BRAF- and multikinase inhibitor, sorafenib, in thyroid carcinoma cell lines of various histological subtypes with and without activating reported . The HThs in FTC ,10,12,49BRAFV600E mutations had the lowest IC50 values for sorafenib, while a slightly higher IC50 value was calculated for the TPC1 papillary cell line, which harbors no BRAFV600E mutation, but the RET/PTC1 rearrangement [BRAFV600E mutations [Proliferation of all cell lines was inhibited by sorafenib within the 48 h treatment period. To our knowledge, ours is the first report about the inhibitory effects of sorafenib not only on cell lines derived from PTCs and ATCs, but also from FTCs. It is in good agreement with recent clinical findings in the phase III DECISION trial of sorafenib in patients with iodine-refractory thyroid cancer, where positive effects of sorafenib on progression-free survival was found in all clinical and genetic biomarker subgroups . In contangement . Cell liangement . IC50 vautations . IC50 vautations  and melautations ,54 treatBRAFV600E mutations: In the fast growing C643 and FTC238 cells, significant effects on cell number were achieved with 0.01 μM. In papillary TPC1 cells significant effects were achieved with 0.05 μM sorafenib while in BHT101 and B-CPAP cells significant inhibition was achieved with 1.0 μM as the lowest concentration. The molecular reasons for these effects are unclear, and may stem from the multikinase-inhibitor activity of sorafenib. It also points to positive effects that may be achieved by sorafenib even in low concentrations due to side effects during sorafenib treatment.The lowest sorafenib concentration that led to a significant antiproliferative effect in our study was the lowest in three cell lines without BRAFV600E mutation. We detected increases in the percentage of cells in subG1 for all cell lines, and different influences on cell cycle progression depending on the cell line. Sorafenib induced a larger proportion of cells of papillary and anaplastic cell lines to enter subG1 than of follicular cell lines, indicating that sorafenib has a different kind of intracellular effect on DNA fragmentation in follicular cell lines. Analysis of the proportion of the treated culture that did not enter subG1 revealed that G1 arrest was induced in all PTC and two of four ATC cell lines, while S phase arrest with G1 decrease was induced in one ATC and three FTC cell lines. The TT2609 follicular cell line showed no alteration in cell cycle phases of the living proportion of the culture. These data concerning the G1 arrest together with the occurrence of a subG1 peak are in agreement with literature data on the TPC1 papillary thyroid carcinoma cell line, the TT medullary thyroid carcinoma cell line [BRAFV600E mutation and HTh7 cells arrested in G1 after sorafenib treatment, one may speculate that inhibition of the overactivated RAF-MAP kinase pathway in these cells contributes to the G1 arrest while other, yet unidentified, molecular effects lead to arrest in the S or G2/M phases in the other cell lines.Sorafenib induced cell death in all 12 thyroid carcinoma cell lines investigated here, regardless of histological derivation or the presence of the activating ell line . On the ell line . These rBRAFV600E mutation, ML1 as a FTC cell line, SW1736 as an ATC cell line harboring the BRAFV600E mutation and HTh7 as an ATC cell line with wildtype BRAF. All four cell lines showed marked LDH release into the medium after 14 and 24 hours of treatment, confirming plasma membrane breakdown and release of cytoplasmic contents. Apoptotic cell death was confirmed by the increased activity of caspases 3 and 7 in all four cells lines. Interestingly, values for LDH release and caspase activities were in the same magnitude in all four cell lines. LDH release was slightly, but not significantly higher in SW1736 and ML1 cells compared to BHT101 and HTh7 cells. In contrast, caspase 3 and 7 activities were slightly, but not significantly elevated in BRAFV600E mutation-positive SW1736 and BHT101 cells compared to HTh7 and ML1 cells. These results are in contrast to recently reported results by Preto and coworkers [BRAFV600E mutation (8505C cell line), but not in thyroid carcinoma cells with wildtype BRAF (C643 and TPC1 cell lines). Preto et al. used the TUNEL assay to quantify apoptosis, and since TUNEL detects DNA fragments directly, it corresponds methodically to quantification of the subG1 peaks in our study. Kim et al. [BRAFV600E mutation in five anaplastic thyroid carcinoma cell lines treated with sorafenib [BRAFV600E mutation to apoptosis resistance, Lee et al. recently showed in the nontransformed PCCl3 rat thyroid cells and in the cervical carcinoma cell line, HeLa, that transfection with an inducible BRAFV600E construct mediates resistance to mitochondrial-induced apoptosis following sorafenib treatment [BRAFV600E mutation on apoptosis induction appears to be different in various cellular contexts. In our experimental setting, apoptosis induction and membrane disruption after sorafenib treatment was not significantly influenced by the histological origin of and BRAF mutational status of thyroid carcinoma cells.We further characterized the mechanism of cell death in detail in four thyroid carcinoma cell lines. We chose BHT101 as an example of a PTC cell line with a heterozygous oworkers , who repm et al.  on the oorafenib  which isorafenib ). Concerreatment . OverallBRAFV600E mutation. Screening of receptor tyrosine kinase receptor activation to identify the inhibitory mechanism of sorafenib exhibited similar results in all four cell lines. Sorafenib inhibited phosphorylation of VEGFRs and PDGFRs receptors, but did not affect phosphorylation of insulin receptors, IGF1R and the EGF family of receptors in thyroid carcinoma cells. These results fit well with results reported for other cell types [V600E kinases, but has no significant inhibitory effects on MEK, ERK, AKT and other signaling pathways [We also examined the of sorafenib on phosphorylation of specific tyrosine kinase receptors in selected thyroid carcinoma cell lines to better assess the impact of differing cellular backgrounds from histological derivation and the presence of the activating ll types ,58. Sorall types . In vitrpathways . Complexpathways -26 can epathways . We obsepathways ). The dipathways . An inhipathways . JNK phopathways .BRAF mutation, these in vitro data are in accordance with data collected so far from patients with various thyroid carcinoma subtypes treated with sorafenib in clinical trials. Our data support the hypothesis that sorafenib may be effective against dedifferentiated thyroid cancers of all histological subtypes and regardless of their BRAF status. The intracellular targets of sorafenib in thyroid carcinoma cells described in this study may allow the development of more specific therapeutic intervention with less side effects.In summary, our results demonstrate that the BRAF- and multikinase inhibitor, sorafenib, exhibited various inhibitory effects on intracellular signaling pathways in thyroid carcinoma cells and caused cell death and cell cycle arrest. Since sorafenib was effective against all thyroid carcinoma cell lines, independent of their histological derivation or the presence of an activating


Reliable identification of different melanoma cell lines is important for many aspects of melanoma research. Common markers used to identify melanoma cell lines include: S100; HMB-45; and Melan-A. We explore the expression of these three markers in four different melanoma cell lines: WM35; WM793; SK-MEL-28; and MM127. The expression of these markers is examined at both the mRNA and protein level. Our results show that the metastatic cell line, MM127, cannot be detected using any of the commonly used melanoma-associated markers. This implies that it would be very difficult to identify this particular cell line in a heterogeneous sample, and as a result this cell line should be used with care. Melanoma is an aggressive form of skin cancer that has the highest incidence rate in Australia2A range of melanoma-associated markers are used to identify different types of melanoma cell lines5MM127 is a metastatic melanoma cell line of human origin, isolated in 1970in vitro monoculture circular barrier assay14Some of our previous work involves investigating how the balance of the rate of cell migration and the rate of cell proliferation affects the collective spreading of a population of MM127 melanoma cellsThis work is organised in the following way. In the Results section we describe the outcomes of three different experimental techniques for identifying different melanoma cell lines using S100, HMB-45, and Melan-A. These techniques include immunofluorescence, Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. These techniques are applied to four different melanoma cell lines, and the results for the metastatic melanoma cell line, MM127, are not as expected. We find that this cell line is not identifiable using any of the three markers.All melanoma cell lines: WM35; WM793; SK-MEL-28 and MM127, are validated using short tandem repeat (STR) profiling . The STR profiling results confirm that all these melanoma cell lines are identical to the reference samples held at Cell Bank. Therefore, all melanoma cell lines in this study are identical to the reference samples held at Cell Bank.http://www.dsmz.de/fp/cgi-bin/str.html) to give the closest match to each cell line we consider. The results from this analysis confirm that WM35, WM793 and SK-MEL-28 are as expected , HMB-45 (27 and 100 kDa), and Melan-A (18 kDa) proteins in the WM35, WM793, SK-MEL-28, MM127 and HaCaT cell lines are analysed using Western blots. The expression of these markers is also examined in primary cells, including keratinocytes and fibroblasts, showing that both keratinocytes and fibroblasts are negative for S100, HMB-45 and Melan-A . The expIn the SK-MEL-28 and WM35 cell lines, HMB-45 is detected as two bands, which is consistent with previous resultsRPL32).The presence of genes that encode specific proteins associated with melanoma cell lines are quantified using qRT-PCR. The HaCaT cell line is used as a negative control because this cell line does not express any melanoma-associated markers as observed in immunofluorescence , WesternS100B), Melan-A (MLANA) and HMB-45 (PMEL) in MM127 is very similar to the negative control [The qRT-PCR results indicate that the expression of S100 ( control . The genMITF); and tyrosinase (TYR)MITF and TYR in the MM127 cells are very similar to the negative control  containing 20% Leibovitz L-15 medium , 4% Foetal Calf Serum (FCS) , 7.5% w/v sodium bicarbonate (Life Technologies), 5 μg/ml insulin (Sigma Aldrich), 1.68 mM calcium chloride, 50 U/ml of penicillin and 50 μg/ml of streptomycin (Life Technologies). Melanoma cell lines MM127 and SK-MEL-28 are maintained in RPMI1640 medium (Life Technologies) supplemented with 10% FCS, 2 mM L-glutamine (Life Technologies), 23 mM HEPES (Life Technologies), 50 U/ml of penicillin and 50 μg/ml of streptomycin. The HaCaT cell line is cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) with 10% FCS and 50 U/ml of penicillin and 50 μg/ml of streptomycin. All cells are routinely screened for 2 and 95% air.Human keratinocytes are isolated from skin discards collected after abdominoplasty and breast reduction surgery. All skin collections are obtained with written informed patient consent. All experimental protocols used in this study were approved by the Queensland University of Technology (QUT) research ethics committee  and the St Andrew’s Hospital ethics committee . The experimental methods carried out in this study are in accordance with the approved guidelines. Keratinocytes are collected from the underside of the epidermis and papillary side of the dermis from surgical skin discards that are placed, overnight, in 0.25% trypsin (Life Technologies) diluted in a 1:1 ratio with phosphate buffered saline (PBS) (Life Technologies). Keratinocytes are grown in a 2:1 ratio on an irradiated feeder layer of 3T3 (i3T3) in full Green’s medium containing DMEM with Ham’s F12 (Life Technologies) in a 3:1 v/v ratio, 10% FCS, 2 mM L-glutamine, 50 U/ml of penicillin, 50 μg/ml of streptomycin, 180 mM adenine (Sigma Aldrich), 1 μg/ml insulin, 0.1 μg/ml cholera toxin (Sigma Aldrich), 0.01% non-essential amino acid solution (Life Technologies), 5 μg/ml transferrin (Sigma Aldrich), 0.2 μM triiodothyronine (Sigma Aldrich), 0.4 μg/ml hydrocortisone (Sigma Aldrich) and 10 ng/ml human recombinant EGF (Life Technologies). Cells are cultured at 37 °C, in 5% CO2 tissue culture flasks  with 254 Medium (Life Technologies) together with Human Melanocyte Growth Supplement (Life Technologies). Epidermal cells, at a density of 8 × 104 cells/cm2, are seeded and incubated at 37 °C in 5% CO2 and 95% air. If the melanocyte culture is contaminated with fibroblasts it is treated with 100 μg/ml of Geneticin® (Life Technologies) for 2–3 days.Melanocytes are grown from an epidermal cell suspension (keratinocyte cell suspension). Isolated epidermal cell suspensions are seeded into T25 cm2 and 95% air for 24 hours. The dermal cell solution is centrifuged at 212 g for 10 minutes and the cells are seeded into T75 cm2 flasks (Nunc®) in DMEM with 10% FCS, 2 mM L-glutamine, 50 U/ml of penicillin and 50 μg/ml of streptomycin at 37 °C in 5% CO2 and 95% air.The dermis, obtained after keratinocyte isolation, is finely minced and placed in a 0.05% collagenase A type I (Life Technologies) solution prepared in DMEM at 37 °C, in 5% CO® 480-1:400 and Alexa Fluor 555®-1:400) (Life Technologies) for an hour. Before proceeding with our experiments, we perform a series of experiments to determine the optimal dilution for each antibody. The optimal dilution was determined by starting with the manufacturer’s recommended dilution and then increasing the dilution to find an optimum result. Cells are washed three times with 0.5% BSA, the nucleus is stained with DAPI – 1:1000 (Sigma Aldrich) and f-actin is stained with Alexa Fluor® 488-1:200 (Life Technologies) for 10 minutes. Coverslips are mounted on glass slides using ProLong® Gold Antifade mountant (Life Technologies).Cells are grown on glass coverslips and fixed with 4% paraformaldehyde  for 30 minutes at room temperature. Cells are permeabilised with 0.1% Triton-X in PBS for 10 minutes, washed with 0.5% w/v bovine serum albumin (BSA) (Life Technologies) in PBS and the non-specific binding sites are blocked using 0.5% BSA for 30 minutes. This is followed by the addition of primary antibody   on cells for an hour, and the secondary antibody (Alexa Fluorg for 15 minutes at 4 °C. The proteins are separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes . Membranes are blocked in Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% skim milk (TBST/milk 5%). All additional immunostaining washes are performed using TBST at room temperature. The corresponding primary antibody is used to incubate membranes overnight at 4 °C; (S100-1:2000 (Dako), HMB-45-1:100 (Thermo Fisher Scientific) and Melan-A-1:1000 (Dako)) in TBST containing 5% BSA (TBST/BSA 5%), and with a secondary antibody, horseradish peroxidase-conjugated anti mouse (1:5000) ; anti-rabbit (1:5000) ; in TBST containing 5% skim milk for 1 hour. We use GAPDH (1:100)  as an internal loading control. These optimal dilutions for each antibody are determined with a series of dilution assays. Membranes are washed five times in TBST and developed with enhanced chemiluminescence, ECL solution . Images are compiled using Adobe Illustrator® and the auto colour balance is adjusted in all figures.Melanoma cell lines  and non-melanoma cells  are lysed by adding lysis buffer containing 80% Radio Immuno-Precipitation Assay (RIPA) buffer , 10% protease inhibitor  and 10% phosphatase inhibitor (Thermo Fisher Scientific). Cells are collected in Eppendorf tubes, vortexed every 5 minutes for half an hour and passed through a 27.5 gauge needle three to four times. Cell lysates of keratinocytes are obtained following removal of the feeder layer. The cell pellet is washed twice with PBS, and the cells are lysed by adding the lysis buffer. Cell lysates are centrifuged at 18000 ® III First-Strand (Life Technologies). The qRT-PCR is performed using an ABI Prism 7500 Sequence Detection System  and SYBR Green (Life Technologies). PCR cycle parameters are the same for all primers: 40 cycles of denaturing at 94 °C for 30 seconds, annealing at 60 °C for 40 seconds and extension at 72 °C for 50 seconds. To quantify the expression of mRNA in each cell line the cycle threshold (Ct) value of each cell line is subtracted from the corresponding Ct value for the internal control, RPL32. The main results are presented here [Total cellular RNA is extracted from melanoma  and non-melanoma cell lines (HaCaT) using Trizol Reagent (Life Technologies) following the manufacturer’s protocol. One μg of RNA is reverse transcribed and cDNA is synthesised using SuperScriptted here  and addiHow to cite this article: Haridas, P. et al. Standard melanoma-associated markers do not identify the MM127 metastatic melanoma cell line. Sci. Rep.6, 24569; doi: 10.1038/srep24569 (2016).


Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA).Macrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Human peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay.In vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected.in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future. Our data contribute to an understanding of monocyte function and macrophage polarization in vitro and of the possibility to influence their behaviour by triterpene containing mistletoe extracts.In our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. Moreover, we demonstrated that VALE shows slight immunomodulatory effects on tumour cell co-cultured macrophages and modulates monocyte chemotactic transmigration  It haenotype. -13. Thusin vitro and in vivo [Natural products from plants became focus in search of new therapeutic options for combating tumours in a multimodal therapy concept ,14,15. A in vivo -20.Viscum album L. (European white-berry mistletoe) are widely used in complementary cancer treatment [in vitro and in vivo [Viscum album L. enhanced the release of pro-inflammatory cytokines and induced anti-apoptotic effects on human isolated peripheral blood monocytes in vitro [Aqueous extracts of reatment , containreatment . Besidesreatment ,23. Due reatment . However in vivo ,22,25-28in vitro .in vitro study was to investigate the effects of soluble tumour cell factors on polarization and chemotaxis/migration of monocytes and macrophages. Additionally the modulatory potential of VALE and its predominant triterpene oleanolic acid on these phenotypic and functional properties was evaluated.The purpose of the present 2 extraction. OA (purity > 98%) was purchased from Sigma-Aldrich . Both reagents were dissolved in DMSO  prior to each experiment. Final concentrations of DMSO in all assays never exceeded the concentration of 1%.Viscum album lipophilic extract (VALE), containing 9.3% oleanolic acid (OA) was kindly provided by Hiscia Institute, Verein für Krebsforschung, Arlesheim, Switzerland and obtained by ultra critical COPossible participation of contaminating mistletoe lectins and viscotoxins in VALE can be excluded, as both toxic proteins are insoluble in lipophilic solvents. In addition the heat labile mistletoe lectins  and viscThe study was approved by the Ethics Committee of Basel, Switzerland (EKBB). Blood samples were taken from 11 healthy donors after obtaining written informed consent. Venous blood was drawn into Li-Heparin tubes  and PBMC were isolated by standard ficoll-paque gradient centrifugation according to manufacturer´s protocol . CD14 positive monocytes were obtained by MACS (Magnetic Activated Cell Sorting) separation technique  according to the manufacturer´s instructions. In brief, PBMC were magnetically labelled with CD14 microbeads, applied to a positive selection column and placed in the magnetic field of a QuadroMACS™ separation unit . After detachment from the cell separator, CD14+ cells were eluted and re-suspended in medium . Purity and viability of the isolated cells were validated using FACS analysis prior to each assay. The purity of isolated CD14+ monocytes was >95% (data not shown).Human breast carcinoma cell lines MCF-7 and HCC1143, pancreas adenocarcinoma cell line PA-TU-8902, prostate >carcinoma cell line DU145 and lung carcinoma cell line NCI-H460 were obtained from DSMZ .In general RPMI 1640 medium, supplemented with 2 mM L-Glutamine and 1% Penicillin-Streptomycin and FCS (either 10% or 1% according to the assay) was used for monocyte/macrophage, NCI-H460, HCC1143 and DU145 cell culture. For MCF-7 cells we used Eagle´s MEM, supplemented with 10% FCS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin and 1 mM Sodium Pyruvate and for PA-TU-8902 cells Dulbecco´s MEM High Glucose supplemented with 1 mM Sodium Pyruvate, 2 mM L-Glutamine, 1% Penicillin/Streptomycin and either 10% or 1% FCS was used .2 and 95% humidity), tumour cell conditioned media were obtained by 24 h incubation of 90% confluent tumour cells in 10 ml of fresh culture medium in 25 cm2 cell flasks. Cell free supernatants were carefully harvested, filtered through 0.5 μm filters and stored in aliquots at −80°C.Cell lines were kept under standard culture conditions  . The cells were incubated at 37°C, 5% CO2 for 10 days, after 3 days and 6 days, medium was partially replaced by fresh medium.Monocytes were differentiated and polarized according to established protocols -34, with5 cells/ml/insert. After 3 days of polarization the media of M2 and tumour cell polarized MDM were replaced by RPMI, either medium alone as control, or in combination with VALE 25 μg/ml or OA 2.5 μg/ml. M0 and M1 media were replaced by RPMI only. The cells were incubated for 3 more days before supernatants were collected and stored at −20°C until further analysis. Cells were trypsinized, washed and analysed by flow cytometry. Five independent experiments were evaluated.On day 10, differentiation medium was replaced by polarization media. 20 ng/ml IFN-γ and 100 ng/ml LPS were used to generate M1 and 20 ng/ml IL-4 and 20 ng/ml IL-13 to generate M2 polarized macrophages . Cells treated with medium only were declared as M0. For the polarization of MDMs by tumour cells we used an indirect co-culture assay. 1 ml/well RPMI was added to the MDM containing wells. Transwell permeable inserts with a pore size of 0.4 μm  were placed into the wells and tumour cells (NCI-H460 or MCF-7 respectively) were seeded at a density of 1.5 × 10Measurement of cell surface marker expression  and viability was performed by four-colour flow cytometry  using specific antibodies for CD11b, CD14, CD36, CD206, CD40, HLA-DR and 7-AAD . Supernatants were analysed for TNF-α, IL-6 and IL-10 in an automated immunoanalyzer .2. The fluorescent cells were washed and re-suspended in medium (1% FCS) at a density of 0.75 - 1,8 × 106/ml. 50 μl of the cell suspension were added onto the polyethylene terephthalate (PET) membrane with 8 μm pores of FluoroBlock Multiwell inserts that was previously coated with 5 μg/well human fibronectin (hFN) . Cells were allowed to rest for 15 min. Into a separate 96-square well flat bottom plate , 200 μl/well of the reagents were added, either 25 nM MCP-1 alone as positive control or in combination with VALE 50 μg/ml or OA 5 μg/ml respectively, medium with 1% FCS was used as negative control and wells with medium without cells were used as blank. The multiwell inserts, containing the calcein labelled cells, were gently lowered into the reagent containing plate. Fluorescence was measured immediately and in 20 minutes intervals afterwards up to 5 hours. Measurements were performed with a bottom fluorescence plate reader (PerkinElmers Victor2 1420) at 485/530 nm (Ex/Em) wavelengths. Between measurements the plate was carefully stored at 37°C, 5% CO2. Four independent experiments performed in duplicates were evaluated. After subtraction of the blank, data were referred to the MCP-1-free negative control. For each replicate, a mean fluorescence value was calculated based on data obtained within 4 hours.The effect of VALE and OA on transmigration/chemotaxis of human peripheral monocytes was investigated using FluoroBlok™ 96-Multiwell Insert Plates . Freshly isolated monocytes were labelled with 1.5 μM Calcein AM  in RPMI-medium (10% FCS) for 30 min at 37°C, 5% COIn a second step we examined the chemotactic ability of tumour cell conditioned media (CM) on monocytes. CMs of the tumour cell lines NCI-H460, HCC1143, DU-145 or PA-TU-8902 were thawed and 200 μl were applied to the respective well in duplicates. All tumour cell CMs were tested alone and in combination with VALE 50 μg/ml or OA 5 μg/ml respectively. The experimental steps were done as described above. Three to five independent experiments were performed for each cell line.All experimental data were evaluated by analysis of variance (ANOVA) with α = 0.05 and type VI decomposition. Pairwise comparisons were evaluated by the LSD-test only if the preceding global F-test was significant (protected Fisher’s LSD-test). This procedure yields a good security against false-positive as well as false-negative results . All calin vitro differentiation of human monocyte derived macrophages by M-CSF and the following polarization into M1- and M2-like cell types revealed specific phenotypic patterns. M1 macrophages significantly enhanced the relative expression of CD40 (p < 0.001) and IL-6 (p = 0.019) and M2 macrophages enhanced the relative expression of CD11b (p < 0.001), CD36 (p = 0.008), CD206 (p < 0.002) and HLA-DR (p = 0.028) compared to the opposite polarization phenotype. CD14 and TNF-α did not significantly differbetween M1 and M2  compared to M1, M2 and MCF-7 polarized macrophages. CD40 expression was significantly lower compared to M1 (p < 0.001), and expression of CD206 and HLA-DR was significantly lower compared to M2 (p < 0.01).MCF-7 co-cultured macrophages exhibited significantly higher levels of CD36 p < 0.001) and significantly lower levels of CD40 (p < 0.001) and IL-6 (p < 0.01) than M1. Expression of CD14 (p < 0.05) was significantly higher and CD206 (p < 0.01) and HLA-DR (p < 0.001) was significantly lower than in M2-like macrophages , an effect that was seen in NCI-H460 polarized macrophages as well (p < 0.05). In NCI-H460 polarized macrophages we observed a trend to inhibition of IL-6 release by VALE. Compared to control, TNF-α production was significantly higher in cells treated with VALE and OA (p < 0.05) Figure . MarkersTo study their effect on cell function we investigated the chemotactic potential of several tumour cell conditioned media on the transmigration of monocytes. As chemotactic control we used 25 nM MCP-1. The migratory activity of monocytes in response to MCP-1 was increased about 3-fold compared to spontaneous migration of monocytes in medium alone (p < 0.001). For all cell lines tested monocyte migration was significantly increased compared to medium alone  on monocytes was shown. Viscum album lipophilic extract and its predominant triterpene oleanolic acid had minor effects on polarization but significantly inhibited MCP-1 induced monocyte transmigration.In our Macrophages display considerable functional plasticity and immediately respond to changes in their microenvironment ,13. Basen vitro treatment of macrophages differentiated from THP-1 human monocyte cell line and MDM with primary tumour cell culture or tumour cell line supernatants [in vivo in histologically normal tissue adjacent to breast tumours [in vitro tumour cell induced differentiation of M2 macrophages showed IL-6 dependent differentiation. Furthermore, increased myelomonocytic markers like CD14 and CD163 were detected after macrophage differentiation by soluble mediators of non-small cell lung carcinoma cell lines [In our experiments tumour cell co-cultured macrophages displayed both M1 and M2 like characteristics and differed between the two tumour cell lines. Such dual M1/M2 phenotypic patterns have been detected previously for example after irnatants  and in v tumours . We also tumours ,43. Thisll lines ,45.in vivo tumour microenvironment, such a macrophage phenotype displaying pro- as well as anti-inflammatory features could occur in tumours. Macrophages within tumours are called TAMs (Tumour Associated Macrophages) and have often been associated with an M2 phenotype. However, this may not always properly reflect the real situation as the tumour milieu itself changes during the development of cancer. Depending on the specific tumour microenvironment, TAMs can exert pro-tumorigenic functions such as metastatic seeding, tumour growth and angiogenesis as well as tumour suppressing activity [Although it is well known that homogenous cancer cell lines strongly differ from primary cultures and both do not fully reflect the interaction and regulation mechanisms of activity ,46,47.in vitro investigations as only a part of the more complex in vivo situation, it still represents important information about the whole framework of cancers and their microenvironment.Taking the results from Based on our previous experience with immunomodulatory effects of triterpene containing Viscum album lipophilic extract on monocytes we were interested if it exerted a modulatory potential on macrophage polarization as well. For this purpose we used concentrations of VALE and OA that were established in prior studies on PBMC, monocytes and fibroblasts and proved to be effective but not cytotoxic ,49. In oin vitro [in vitro chemotactic activity of monocytes and there seemed to be a correlation between chemotactic activity and tumour-associated macrophage content in ovarian cancer [in vitro chemotactic potential for supernatants of the four tumour cell lines we used. As Wang et al. found chemotactic as well as anti-chemotactic regulation mechanisms of supernatants from human sarcoma and ovarian carcinoma cells [As tumour cells are known to possess the ability to recruit immune cells, especially monocytes into their microenvironment where they differentiate into TAM´s, we investigated the effect of supernatants from different tumour cell lines on the migration of monocytes in a transwell chemotaxis assay. As positive control for chemotaxis we used MCP-1, that is also referred to as chemokine (C-C motif) ligand 2 (CCL2) and possesses chemotactic activity for monocytes, macrophages and T-lymphocytes. In our study monocytes were attracted by supernatants of tumour cell lines, suggesting soluble factors derived from tumour cells to mediate migration. Our findings are in line with other investigations on the chemotactic potential of tumour cell supernatants on monocytes. Supernatants of colon, breast cancer or myeloid leukemic cell lines were shown to induce significantly different migratory activity of monocytes in vitro ,56. Supen cancer . Accordima cells , the difin vitro [VALE and OA both significantly decreased the migration of monocytes towards MCP-1 in doses not toxic for these cells. MCP-1 is one of the most prevalent cytokines in tumour microenvironment and has been suggested to be one of the principle determinants of human tumour macrophage content , mediatiin vitro , a findiVALE and OA did not have an impact on monocyte migration induced by tumour cell supernatants, giving the assumption that other factors besides MCP-1 influence the attraction of these cells and that the impact of VALE and OA on monocyte migration is somehow correlated with MCP-1 effects. It is already known that soluble factors besides MCP-1 are able to influence monocyte migration. For example, Clahsen et al. showed induction of mouse monocyte migration via IL-6 .Further research on possible mechanisms of VALE- and OA-induced modulation of cell migration could lead to a better understanding of the therapeutic options regarding these triterpenes.in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future.Our results demonstrated that co-culture with two tumour cell lines might result in a dual macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. The observed chemotactic potential of tumour cell conditioned media in our study could serve as a model for further research on modulating tumour cell induced monocyte migration. The triterpene containing compound VALE was shown to modulate monocyte chemotactic transmigration and have some immunomodulatory effects on tumour cell co-cultured macrophages


Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly  as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly.  Glossina sp.). Therefore understanding the factors that allow for the successful infection of the insect vector remains a relevant research interest. The fly is more than a passive vector as the parasites undergo specific development programmes before becoming infective again. These can be relatively simple as in the case of Trypanosoma vivax that is mostly found in the mouthparts and alternates between two major forms. However, this is much more sophisticated in the Trypanosoma brucei subgroup , where infection is established in the posterior midgut and proceeds anteriorly towards the proventriculus to culminate in the salivary glands, giving rise to metacyclic trypomastigotes that are the only parasites infective to mammalian hosts [T. congolense that proliferates first in the gut and then reaches the proboscis before attaching to the surface of the labrum and releasing infective cells in the hypopharynx [Trypanosomes are the causative parasites of human African trypanosomiasis, also called sleeping sickness. Although on the decline ,2, the han hosts ,4. An inopharynx . To survopharynx .T. brucei during its cyclical development.Trypanosomes are members of the Excavata, one of the major early branching eukaryotic lineages. Their morphology is characterised by an elongated shape and the presence of a flagellum attached to the cell body. The flagellum is a multifunctional organelle, with known roles in motility, morphogenesis and attachment to the epithelium of the salivary glands (review in ). It hasT. brucei [Paramecium caudatum cilia to ADP decreases beat frequency, but is restored upon addition of phosphorylated arginine [bona fide energy shuttle in flagellated protists, generation and access to phosphagens could be important in maintaining the beat frequency of spermatozoa and protist flagella.A proteomics analysis of intact flagella purified from cultured procyclic trypanosomes revealed the presence of the enzyme arginine kinase (AK) in the membrane and matrix fraction . The enz. brucei . The pre. brucei ). In bot. brucei ,17. Whilarginine . This arAK genes were likely acquired by horizontal gene transfer from an invertebrate host and that separate duplication events led to the emergence of flagellar forms of AK in T. brucei and possibly in T. congolense, the two species that undergo the most exhaustive development in the tsetse fly. The flagellar AK is expressed in all stages of development but it appears more abundant in insect stages compared to the bloodstream form. Knockout of the flagellar AK in T. brucei mildly affects parasite motility but leads to lowered infectivity and competitiveness in tsetse, supporting a contribution to infection. To our knowledge, this is the first in vivo evidence regarding the possible relevance of flagellar AK in the life cycle of T. brucei. Finally, we speculate about the possible role of AK3 in trypanosome development and about its mechanism of flagellar targeting.Here we investigated the possible role of arginine kinase during parasite development in the tsetse fly. We present phylogenetic data revealing that trypanosome This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the European Union (European Directive 2010/63/UE) and the French Government. The protocol was approved by the “Comité d’éthique en expérimentation animale (CETEA) de l’Institut Pasteur” N°89 (Permit Number: 2013–0128 Blisnick). All efforts were made to minimize suffering.AK copies in T. brucei and T. congolense, further analysis was restricted to the trypanosome only sequences, and performed on nucleotides. A maximum likelihood tree was obtained by PhyML, the GTR model and 100 boostrap replicates of the original alignment.Homologues of TbAK were exhaustively searched by Blastp in current sequence databases. Multiple protein alignments were performed with Muscle , and triT. b. brucei AnTat 1.1 (wild-type or WT) cell line [SDM-79 + Glycerol). Cell concentration was determined using the Z2 cell counter (Beckman Coulter). Bloodstream AnTat 1.1 trypanosomes were cultured in Creek’s minimum medium [2 and allowed to attain 2 X 106 parasites/ml culture prior to harvest for transformation to the procyclic stage, IFA or western blots for the comparison of AK3 amounts between bloodstream and procyclic trypanosomes. For transformation, bloodstream trypanosomes were suspended in DTM medium with 3 mM isocitrate/cis-aconitate and cultured at 27°C for 24 h [Cell lines for the investigation were generated from the tsetse-transmissible ell line . Procyclell line  supplemeell line  or the blasticidin (BLA) drug resistance gene flanked by upstream and downstream regions of the AK3 gene  were chemically synthesized and cloned in a pUC57 plasmid . PCR amplicons were generated with primers F1: TTTGGCAAATGCTCACAAGA and R1: TCTATACACAAGTACCTCCC (Eurogentec) which anneal to the sequences 300 bp upstream of the AK3 start codon and downstream of AK3 stop codon, respectively. Amplicons with either the blasticidin resistance gene (999 bp) or the puromycin resistance gene  were gel-purified (Macherey-Nagel) prior to transfection. Nucleofections were carried out using the Amaxa nucleofactor (Lonza) and clonal populations were obtained with sub-cloning by limiting dilution. Generation of the ∆ak3 cell line was accomplished by 2 successive rounds of nucleofection to replace both alleles of endogenous AK3 in WT with BLA and PURO genes. For the purpose of generating an add-back cell line (AB), the AK3::Ty1 gene was synthesized and integrated into the pHD430 plasmid [AK3, the 30 nucleotides encoding the Ty1 sequence (GAGGTCCATACTAACCAGGACCCACTTGAC)[AK3 including the stop codon. This plasmid was linearized with the NotI restriction enzyme prior to nucleofection for integration into the ribosomal intergenic spacer in the ∆ak3 cell line. An additional round of nucleofection was carried out on clonal AB cell line with the pHD360 vector [AK3 in the genome of the transformed cells, genomic DNA was isolated [AK3.For the purpose of generating a cell line deprived of AK3, we used a double knockout approach. Sequences containing either the puromycin (CACTTGAC) and the 0 vector . As the 6 parasites/ml  as a first meal through a silicone membrane. Flies were fed twice a week with defibrinated sheep’s blood and starved for at least 48 h prior to dissection. Dissections were carried out on glass slides in PBS under a binocular dissection microscope by removing the alimentary canal of the tsetse from the hindgut up to the crop and proventriculus to determine the presence or absence of parasites in the various sections . Salivary glands were dissected in a separate drop of PBS on the glass slide. Infection data from dissection experiments were only considered for statistical analysis in biological replicates where more than 10 individual insects were left alive in all experimental groups at the time of dissection. For parasites meant for IFA analyses, the whole gut was torn apart with forceps and the parasites were transferred into an Eppendorf on ice, while individual sections of the alimentary canal were removed and dropped directly into separate Eppendorf tubes with 100 μl of PBS on ice when checking for the proportion of cells expressing AK3::Ty1. Both samples were subsequently treated with 4% PFA prior to IFA.Teneral males were collected 24 to 48 h post-eclosion in our tsetse insectarium that is maintained at 27°C and 70% hygrometry. Collected flies were separated into Roubaud cages and infected by directly feeding with SDMG medium containing 6–9 X 10Sample preparation for immunofluorescence assays (IFA) was as described in . BrieflyIFA Image acquisition was carried out on a Leica 4000B microscope with a 100 X objective lens using a Hamamatsu ORCA-03G camera. The fluorescence light source was from a Lumencore LED system with the following incident light intensities: Cyan , UV  and Green . Image acquisition was controlled using Micro-manager and images were taken with the min/max threshold set at maximum. Subsequent normalization of signals was carried out by parallel manipulation of min/max signal against controls in ImageJ , and imaFor western blotting, sampled were prepared by washing cells in serum-free medium before dilution with PBS and boiling in Laemmli. SDS-PAGE separation was carried out after loading 10 μg (corresponding to 1 million cells) total cell protein per lane. Proteins were transferred onto polyvinylidene fluoride membranes overnight at 4°C and blocked with 5% skimmed milk in PBS with 0.1% Tween 20 for 1 h prior to probing. The following primary antibodies were used by dilution in 1% milk in PBS with 0.1% Tween 20: L8C4  .6 were maintained in SDMG medium at 27°C prior to video acquisition. Movies  were captured with a 10 X objective lens on a Leica 4000B microscope with a Hamamatsu ORCA-03G camera using HCImage Live software (Hamamatsu). Movie capture was carried out in an enclosed room maintained at 25°C. Videos were converted to MPEG format and analysed using medeaLab CASA Tracking V.5.5 software (medea ABGmbH).Cells at 6–9 X 10One-way ANOVA, non-continuous t-tests, linear regression were carried out on Prism (GraphPad). Differences are considered significant if P < 0.05 (denoted with *) and highly significant if P < 0.001 (denoted with **).AK gene from Trypanosoma cruzi, highlighting the remarkable conservation of the core protein that shows ~70% identity with various insect or crustacean AK [AK genes in all Trypanosoma subspecies investigated to date but not in Leishmania [AK genes were identified according to the species: 4 in T. b. gambiense, 3 in T. brucei and T. evansi, 2 in T. congolense and only 1 in T. vivax or T. cruzi  possess EF-hands and an amino-terminal tail of 24 amino acids that is necessary and sufficient for flagellum location [T. congolense AK protein recapitulates most of these features: amino-terminal positioning, glycine in position 2 and 7 positively charged residues (including 6 lysines).In their targeting studies, Voncken et al. could not identify a conserved domain that would explain the targeting of AK3 into the flagellum . Here, wlocation ,39. Thislocation ,40,41. AFLA1RNAi strain [T. cruzi AK protein [T. brucei by immunofluorescence assay (IFA), a signal that is lost upon AK3 RNAi silencing . In total protein samples  resulted in total loss of the flagellum signal, also confirming the specificity of the antibody [Several lines of evidence suggest that AK3 could perform more significant function(s) during parasite development in the fly: restricted presence in antibody . Neverthantibody . This su∆ak3 cell line with both copies of AK3 replaced by a puromycin and a blasticidin resistance gene was generated. Second, an exogenous Ty1-tagged version of AK3 (AK3::Ty1) was added from a subsequently transfected pHD430 vector [∆ak3 cell line, generating the ∆ak3 + AK3::Ty1 (AB for Add-Back) cell line that constitutively over-expresses TbAK3::Ty1 from the rDNA spacer under the transcriptional control of an EP procyclin promoter. Both cell lines were produced without noticeable problems and were sub-cloned immediately after selection for characterisation at the genomic and protein level. First, genomic DNA was extracted from all three cell lines and PCR was performed using two combinations of primers: one for the upstream and downstream regions the AK3 gene . IFA probing for both AK and Ty1 simultaneously confirmed the loss of flagellar AK in the ∆ak3 cell line, while the AB cell line expressed a flagellar AK that was also Ty1-tagged  background, which is tsetse infective. First, a 0 vector  into theck lines . Westernck lines . In the ck lines . This back lines : by 1.6 1-tagged .AK1-3RNAi cell lines (in the 29–13 background)[∆ak3 cell lines exhibit a slight but significant growth phenotype in culture (∆ak3 (16.7 ± 1.1 h) cell lines , the ∆ak culture . In the ll lines . Despitell lines . This suTi cell line where AK3::Ty1 expression should be inhibited by the tet-repressor in the absence of tetracycline but should be restored in its presence [Ti differed from the AB in that the expression of AK1 and AK2 was not simultaneously increased  but this was not statistically significant. ABTi cells induced with tetracycline for 24 h prior to starting the growth assay negated this growth phenotype, reverting to growth rates close (14.8 ± 1.0 h) to that of the WT cell line  reduction in motility compared to WT cells  was measured at cell densities of 6–9.106 cells/ml as this was the culture density used for tsetse infection experiments. This reduced motility was partially restored in the AB cell line. To evaluate the consequences of absence of AK3 in the knockout cell line and the possible negative effect of overexpression of AK1/AK2, transmission electron microscopy analysis was carried out. Attention was initially focused on the flagellum but no visible structural defects could be detected neither in the 9 + 2 microtubule doublets of the axoneme and its outer dynein arms, nor in the paraflagellar rod and the membrane in transmission electron microscopy images of the WT (n = 33) and ∆ak3 (n = 22) cell lines . We reasoned that the disparity in doubling times may be interfering with any affect AK3 absence/presence may have on tsetse infection rates, and proceeded to infect and score for parasite presence at 20 days or more post infection.We next attempted to determine if absence of AK3 had an effect on tsetse infection. Because of the severe reduction in growth in the AB cell line, initial experiments were carried out and tsetse flies were dissected at 10 days post infection, revealing comparable infection rates for ∆ak3-infected tsetse had neither AK nor Ty1 signal in their flagellum. The majority of parasites extracted from AB-infected tsetse had both an AK signal and a Ty1 signal in their flagellum as expected. Infection with the WT strain resulted in the classic frequency of flies presenting an infection in their midgut (40.4 ± 13.0%). From the same infection experiments, it was observed that tsetse infected with ∆ak3 cells were less likely to be infected when dissected at more than 20 days post infection . The infection rates shown here are for midgut infections only, as infections in the salivary glands were sporadic and low regardless of the cell line and no trend could be discerned from this (data not shown). Infections with the ABTi cell line were also attempted but the presence of tetracycline increased significantly the mortality of flies and hindered data interpretation (data not shown).Immunofluorescence assays with simultaneous probing for AK (anti-TcAK) and AK3::Ty1 (anti-Ty1) in trypanosomes extracted from tsetse in infection experiments revealed that no significant genetic drift occurred in the parasite population during the 20 day incubation period leading up to dissection . The majnfection , with a ∆ak3 and AB parasites and carried out an IFA on parasites extracted from the posterior midgut and anterior midgut. The majority of parasites extracted in these co-infection experiments had a Ty1 signal in the flagellum  possess an amino-terminal tail of 24 amino acids that is necessary and sufficient for targeting to the flagellum [Leishmania, which is also the case of calflagins. Unfortunately, the crystal structure is only known for the T. cruzi AK [Here, we propose that AK3 flagellar targeting is achieved by its N-terminal extension that shares remarkable similarities with the flagellum calcium-binding protein (FCaBP) in lagellum ,39. Firslagellum ,40. Howelagellum  that turlagellum . Moreovecruzi AK  that doeT. brucei and T. congolense have the most complicated life cycle within the tsetse [T. congolense, whereas T. brucei needs to reach the salivary glands to undergo another round of differentiation, proliferation and ultimately production of metacyclic trypanosomes [Amongst African trypanosomes, e tsetse . Upon inanosomes . SurvivaAK3 mRNA levels in the procyclic form is in agreement with an increase in protein expression. This change in expression may reflect the different metabolism of these life cycle stages [AK3 was found to be more highly expressed in procyclic trypanosomes compared to their bloodstream counterparts based on observations using IFA and western blots. This is in agreement with previous findings on the mRNA level using northern blots . As RNA e stages ,50 or co∆ak3 cell line showed a slight impairment in growth rate in vitro that was further exacerbated in the AB cell line. However, no statistically significant difference in growth rate could be observed in the ABTi cell line, suggesting that absence of AK3 is not essential per se in culture conditions for the procyclic form. The only visible phenotype was a slight reduction in motility, without noticeable impairment in base-to-tip or tip-to-base beating of the flagellum. This is likely to be insufficient to perturb cell division, as reported for various RNAi mutants where flagellum motility is reduced but not blocked [What could be the function of AK3 in the flagellum? This organelle is central to the parasite cycle by controlling cell morphogenesis ,51, cell blocked ,58.in vivo where parasites need to migrate from the posterior to the anterior midgut. A previous investigation where parasite forward motility was ablated by suppression of the DNAI1 gene demonstrated that this prevents the migration of trypanosomes from the posterior to the anterior midgut of the tsetse [The situation could be different e tsetse . Here, ae tsetse . OverallWe propose that this slight advantage could result from the functioning of AK as phosphagen kinases that are involved in ATP buffering. For example, creatine kinase is localised in the flagellum of sea urchin sperm to mediate regeneration of ATP during periods of high activity ,17. LittGlossina pallidipes in Tanzania identified up to 40% of dissected fly proboscises to harbour more than one species of trypanosome, while a similar study on Glossina palpalis in Cote d’Ivoire identified mixed infections of T. congolense, T. vivax and T. brucei in field-caught flies [G. m. morsitans have demonstrated that flies with a mature parasite infection are just as susceptible to infection by a second trypanosome species compared to uninfected flies [In the field, mixed trypanosome infections in tsetse are not unusual, whether it is between different trypanosome species or between different subspecies. Molecular identification of trypanosome species from field-caught ht flies ,71. Laboed flies . Overall