N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N2-yl)-4AQO and 3-(deoxyadenosin-N6-yl)-4AQO. This study was designed to assess the shape of the dose–response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine–guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose–response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay.4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts,  Genetic toxicology involves the assessment of a substance’s ability to induce DNA damage, which is an essential consideration for human health risk assessment because DNA damage is an underlying cause of mutations that have the potential to initiate carcinogenesis. It is essential to investigate and understand the biological significance of genotoxic effects of chemicals at the low-dose exposure range to improve human health risk assessment and to establish if DNA reactive compounds follow linear or non-linear dose–response relationships.in vitro testing to ensure that DNA damaging effects were identified and because of the assumption that genotoxins follow a linear relationship that was extrapolated back to the low-dose region -4AQO, 3-(desoxyguanosin-N2-yl)-4AQO and 3-(deoxyadenosin-N6-yl)-4AQO (4-Nitroquinoline 1-oxide (4NQO) is a known mutagen and carcinogen and is therefore used in various genotoxicity assays as a positive control . The cheeactions . The enzeactions . 4NQO aneactions . The acteactions ; of thesyl)-4AQO ,13. The yl)-4AQO . It is kyl)-4AQO . Furtheryl)-4AQO , and it yl)-4AQO ,17.in vitro and to investigate the effects of using different study designs on the points of departure (PoD) and genotoxic potency. Chromosomal damage was investigated using the in vitro micronucleus (MN) assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine–guanine phosphoribosyltransferase (HPRT) forward mutation and comet assays. Comparative studies were performed in two laboratories, Swansea University, Swansea, UK and AstraZeneca, UK.This study was designed to assess the shape of the dose–response curve at low concentrations of 4NQO in human and mouse cells 4NQO was acquired from Sigma–Aldrich  and dissolved in dimethyl sulfoxide . Before use, the chemical was freshly diluted from a stock solution  with DMSO.+/− cell line that constitutively expresses a high level of native CYP1A1 . Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. For MCL-5 cells, hygromycin B  in acetic acid (35mM) was added at each passage to a final concentration of 200 µg/ml to ensure plasmid retention  supplemented with 10% donor horse serum  and 1% etention .l-glutamine , 2mM sodium pyruvate , 200 IU/ml penicillin  and 200 µg/ml streptomycin  at 37°C, 5%CO2. L5178Y cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated donor horse serum, 2mM l-glutamine, 2mM sodium pyruvate, 1% Pluronic F68 , 200 IU/ml penicillin and 200 µg/ml streptomycin at 37°C, 5%CO2.TK6 cells at AstraZeneca were cultured in RPMI 1640  supplemented with 10% heat-inactivated donor horse serum , 2mM in vitro cytokinesis-block micronucleus (CBMN) assay, AHH-1 and MCL-5 cells were treated with 4NQO over a range of concentrations up to 0.7 µg/ml for 4h, followed by one cell cycle of cytochalasin B . TK6 cells were treated with 4NQO over a range of concentrations up to 0.03 µg/ml for 4, 24 or 48h, respectively, followed by one cell cycle of cytochalasin B (4.5 µg/ml). Further, TK6 cells were treated for 24h with 4NQO for the HPRT assay study with 0.0009, 0.003, 0.01 and 0.02 µg/ml 4NQO. Medium was used as negative control, whereas DMSO was used as the solvent control. The experiments were carried out in triplicate.For the in vitro MN assay, L5178Y cells were treated with 4NQO for 4h, followed by 24-h recovery time over a range of concentrations up to 0.05 µg/ml. Further, TK6 cells were treated with 4NQO for 4h plus 40-h recovery period and 24h plus 24-h recovery time over a range of concentrations up to 0.03 µg/ml. For the comet assay, TK6 cells were treated with 4NQO for 3h over a range of concentrations up to 0.06 µg/ml 4NQO because of previous experiments conducted at AstraZeneca (data not shown). The experiments were carried out in duplicate.For the mononucleated The treatment regimes chosen covered a range from producing 55±5% cytotoxicity to little or no toxicity, as higher levels may induce chromosome damage as a secondary effect of cytotoxicity .5 cells/ml for 24h at 37°C, 5%CO2 prior to treatment. Details of the assay are described by Brüsehafer et al.  procedure routinely performed at AstraZeneca used the semi-automated MN scoring system Metafer . Ten-millilitre cell suspensions were seeded at 2×105 cells/ml and immediately treated with 1% v/v solvent control and the test chemical at 37°C, 5%CO2. After the different incubation times, all of the cell suspensions were removed from each flask and transferred into appropriately labelled tubes. The cells were centrifuged , the supernatant was discarded and the pellets washed with phosphate-buffered saline (PBS), before re-suspending the cells in 10-ml fresh media. The cells were cytospun onto polished glass slides. Shandon mega-funnels  were used to harvest cells for the Metafer System. The cells were fixed in 100% methanol for 15min. For the semi-automated scoring protocol, the cells were stained for 10min with a 4′,6-diamidino-2-phenylindole + vectashield  solution and scanned with the Metafer4 software. All experiments carried out at AstraZeneca were run in duplicate. One thousand mononucleated cells were scored per replicate.The Relative population doubling (RPD) was used to measure cytotoxicity and cytostasis in the absence of cytochalasin B. Therefore, satellite cultures were seeded as described previously for the CBMN assay . Cells wRPD was calculated as follows:where5 cells/ml), and the cells were grown for 3 days. After 3 days, the HAT medium was washed off with PBS. Afterwards, the cells were re-suspended in 50-ml fresh medium, and hypoxanthine–thymidine (HT) was added to the culture medium for 24h. After washing off the HT medium with PBS, TK6 cell suspensions were seeded at 1×105 cells/ml for 24h at 37°C, 5%CO2. The cell suspensions were then treated with 4NQO, and after the treatment period, 4NQO was then washed off with PBS, new medium was added and the cells were sub-cultured to 1.2–1.5×105 cells/ml in 50-ml flasks (Day 1). The cells were sub-cultured and left to grow for 13 days in total to enable expression of the HPRT− mutants. Cells were sub-cultured at Days 1, 3, 5, 7, 9 and 11. After the phenotypic expression period at day 13, the cells were added to 96-well micro plates.The first step of the HPRT assay was the mutant cleansing stage to remove background mutants within the TK6 cell population. Therefore, hypoxanthine–aminopterin–thymidine (HAT) was added to the TK6 culture (4–5×107 cells in 60ml were treated with 240 µl of 6-thioguanine (0.6 µg/ml) (6-TK) for selection. Five plates per dose were used with 100 µl of cell suspension in each well. Plates were scored for colony formation after 14 days of continuous incubation at 37°C, 5%CO2. Scoring criteria specified that only colonies with 20+ cells in diameter were scored. Further, it was ensured that separate colonies were clearly apart, taking clonal expansion into account. These experiments were carried using three separate stock solutions, but with the same cell passage.For mutant frequency, TK6 cells with 2.4×10in vitro comet assay, TK6 cells were treated with 4NQO that was then washed off with PBS and the cells were re-suspended in fresh media. The cell number was adjusted to 1.5×105 cells/ml. After that, 1ml of the cell suspensions were sampled into labelled Eppendorf tubes and washed with 1ml cold 1× PBS. After centrifugation, the supernatant was removed from the pellet and the cells were re-suspended with 200-µl low-melting agarose and 3×40 µl drops, resulting in three gels, were added per glass slide (pre-coated with 0.5% agarose) and covered with a coverslip. Slides were then placed on a cold tray, which allowed for gels to set after which the coverslips were removed and the slides were placed in cold lysis solution  in a dark container over night at 4°C. Following lysis, the slides were washed twice for 5min in 1× buffer F  at room temperature. hOGG1 (+hOGG1) or buffer F (−hOGG1) (60 µl) were then added to each slide, topped up with a coverslip and incubated in a humidified box at 37°C for 10min. After removing the coverslips, the slides were placed in an electrophoresis platform and covered with electrophoresis buffer  for 20min at 4°C to allow the DNA to unwind. Afterwards, the electrophoresis was performed at 0.7V/cm, 300 mA for further 20min at 4°C. To neutralise, the slides were removed from the electrophoresis platform and immersed in three changes of neutralisation buffer [0.4M Tris–HCl (pH 7.5)] for 5min each time at room temperature. Finally, the slides were stained with 60-µl propidium iodide (20 µg/ml) for 30min, before scoring using a Comet IV capture system . Fifty nuclei per gel were scored and the tail intensity that is defined as the percentage of DNA migrated from the head of the comet into the tail was measured for each nucleus scored (For the s scored . The expt-test (n = 2) or a one-way analysis of variance approach (n = 3), followed by a two-sided Dunnett’s post hoc test with log-transformed data based on Kolmogorov–Smirnov test for normality and Bartlett’s test for homogeneity of variance .Error bars are represented as standard deviation of the mean. Student’s paired variance , were usin vitro genotoxicity assays because of its known mutagenic and carcinogenic properties. The aim of this study was to investigate the low dose–response relationships of 4NQO in human lymphoblastoid cells to calculate and compare PoDs for genotoxic activity. The chosen exposure and recovery times were optimised using empirical experiments bearing in mind the need to keep cytotoxicity levels below 55±5% as dying cells can confound the MN assay results.4NQO is used as a positive control in various To evaluate if 4NQO requires metabolic activation and to investigate the role of p53 proficiency, the metabolically competent human cell line MCL-5 and the parent cell line AHH-1, which holds some degree of metabolic competence, were treated with 4NQO at Swansea University. Both these cell lines have a heterozygous p53 mutation in codon 282. At AstraZeneca, the mouse lymphoma cell line L5178Y  was treated with 4NQO. L5178Y cells are frequently used for genetic toxicity studies. Short-term treatments with and without prolonged recovery times were used for the investigation. AHH-1 cells treated with 4NQO for 4h over a range of concentration up to 0.7 µg/ml, followed by one cell cycle of cytochalasin B (22h) showed significant increases in MN induction only at 0.3 µg/ml. However, MCL-5 cells showed no significant increases in MN induction for the short-term treatment over the range of concentrations chosen. At 0.7 µg/ml, high toxicity levels were observed in AHH-1 and MCL-5 (61% in AHH-1 and 53.5% in MCL-5) cells . L5178Y supplementary Figure 1, available at Mutagenesis Online). BMD modelling utilises a set of statistical models to estimate a defined increase above its control response values (benchmark response), along with its confidence limits. For the AHH-1 data after treatment with 4NQO, a BMD10 (10% increase above the background) was calculated at 0.1238 μg/ml with confidence intervals between 0.0678 and 0.3007 μg/ml, respectively. No dose response was observed for the MCL-5 data; however, the covariate approach enabled the derivation of BMD10 and BMDL10 metrics at 46.4590 and 0.2120, respectively, along with an infinite BMDU10. The L5178Y data set revealed a BMD10 at 0.0010 μg/ml with a BMDL10 and BMDU10 of 0.0006 and 0.0016 μg/ml, respectively . For the TK6 data after treatment with 4NQO for 4h followed by 18h of cytochalasin B, a BMD10 was calculated at 0.0044 μg/ml with BMDL10 at 0.0025 μg/ml and BMDU10 at 0.0092 μg/ml. TK6 cells treated with 4NQO for 24 and 18h of cytochalasin B revealed a BMD10 at 0.0029 μg/ml with BMDL10 at 0.0019 and BMDU10 at 0.0049 μg/ml, respectively, whereas TK6 cells treated with 4NQO for 48 and 18h of cytochalasin B revealed a BMD10 at 0.0103 μg/ml, BMDL10 at 0.0053 μg/ml and BMDU10 at 0.0597 μg/ml. For the TK6 cells with an extended recovery time of 24h after 24-h 4NQO treatment, a BMD10 was calculated at 0.0057 μg/ml with a BMDL10 and BMDU10 at 0.0034 and 0.0107 μg/ml, respectively. TK6 cells with an extended recovery time of 40h after 4-h 4NQO treatment revealed a BMD10 at 0.0047 μg/ml with a BMDL10 and BMDU10 at 0.0026 and 0.0108 μg/ml, respectively .Subsequently, the dose responses were further assessed by BMD modelling . Further experiments into the endogenous DNA repair protein levels and activity in the different cell lines need to be conducted to investigate the influence of variations in DNA repair on genotoxicity studies between cell lines. In addition, the differences in metabolic proficiencies of the various cell lines did not seem to have an impact on the observed dose responses, as 4NQO showed no increased genotoxicity in the cells with known metabolic competence. The chromosomal damage studies in p53 competent TK6 cells revealed that the short-term treatments (4h) without prolonged recovery time and extended treatments (24 and 48h) showed little to no chromosomal damage induction up to 55±5% cytotoxicity. However, extending the recovery time up to two cell cycles in the mononucleated assay increased the magnitude of MN induction at the higher concentration range. A study from Sobol et al.  showed t  and AstraZeneca.This study was supported by a Collaborative Award in Science and Engineering (CASE) studentship from


The results would be useful for learning the muscle development mechanism in teleosts.Objectives. According to myosatellite cell lines (MSCs) established Paralichthys olivaceus were isolated and cultured in vitro, respectively, and the cells were characterized at the morphology and molecular level; meanwhile, the performance of these cells’ proliferation and differentiation were analyzed.Materials and Methods. The skeletal muscle cells from the diploid and triploid olive flounder S(2n) and POMSCS(3n)) from diploid and triploid flounder have been respectively subcultured for 67 times and 66 times. The cultured cells were mostly spindle-like mononuclear cells. They have normal flounder diploid karyotype (2n=48t) and triploid karyotype (3n=72t), respectively. Muscle satellite cell gene marker (pax7b) and myogenic cell protein marker (Desmin) were all expressed in cells of two cell lines. Both of the cells could differentiate into the large polynucleated muscle fibre cells, and immunofluorescence reactions of myosin heavy chain (MyHC) were positive. There were more cells of POMSCS(3n) to differentiate into the muscle fibre cells than that of POMSCS(2n). However, POMSCS(2n) cells proliferated more rapidly than those of POMSCS(3n) (P < 0.05). The significant fluorescent signals were observed in both POMSCS(2n) and POMSCS(3n) cells after transfected with pEGFP-N3 reporter plasmid.Results. Two new skeletal muscle cell lines (POMSCConclusions. The two cell lines have been established and characterized as MSCs. We suppose that it might be the differentiation capacity, rather than the proliferation activity of MSCs to play a key role in the better growth of triploid ones than diploid. Both cell lines will become the ideal tools to learn the mechanism of fish MSCs proliferation, differentiation and regeneration during muscle development in the future. In vitro, cultured MSCs can realize a complete genesis process: proliferation, differentiation, fusion and the formation of the multinucleated myotubes (Oncorhynchus mykiss)  (Cyprinus carpio) (Sparus aurata) (in vitro system for trout muscle satellite cell culture was established and used to examine the effect of myostatin (MSTN) on proliferation or differentiation of myogenic cells  is a very important tissue for fish swimming and live, and one of the food resources for human. Fish body weight mainly depends on both muscle fibre number increase (hyperplasia) and muscle fibre size increase (hypertrophy) as skeletal muscle contributes to 40–60% in fish body mass . MSCs armyotubes . The resmyotubes . In tele mykiss) , Atlantialar L.) , carp (C carpio) , and gil aurata) . An in vic cells . But comAristichthys mobilis), large yellow croaker (Larimichthys crocea) and turbot (Scophthatmus maximus). Some papers showed that growth of triploid fishes could be more rapid than that of diploid ones  homologues, MSC marker genes, during embryonic development (S(2n) and POMSCS(3n)) respectively derived from the diploid and triploid flounder were established, and on the basis of analyzing their cytological features, the characteristics of these cell proliferation and differentiation were compared.Olive flounder nd so on . The reselopment . As a coelopment , and theelopment . The resAdult flounders weighing about 100–200 g were purchased from Nanshan market , and were temporarily reared in a 500 L aerated seawater tank at the institute aquarium and fed with commercial particle food twice a day.Triploid flounders were artificially induced according to Experiments involving live animals were conducted in accordance with the “Regulations for the Administration of Affairs Concerning Experimental Animals” promulgated by the State Science and Technology Commission of Shandong Province. The study was approved by the ethics committee of Institute of Oceanology, Chinese Academy of Sciences.−1 3-Aminobenzoic acid ethyl ester methanesulfonate  and wiped with 70% (v/v) ethanol. After removal of the skin and the red muscle tissue, 0.4–0.6 cm3 dorsal white muscle tissue was immediately excised and immersed in Minimum Essential Medium  containing 400 U ml−1 penicillin and 400 µg ml−1 streptomycin. The tissue was washed twice with phosphate-buffered saline  and minced finely into small pieces (1 mm3) in a new tissue culture dish. The tissue pieces were suspended with 1 ml of MEM complete medium , 10 ng ml−1 bFGF , 100 U ml−1 penicillin , 100 µg ml−1 streptomycin ) and then the tissue suspension was planted into a 25 cm2 standard cell culture flasks in succession. The suspension was incubated at 25 °C in a CO2 incubator . After 24 h of attachment, 1 ml MEM complete medium was carefully added to the flasks. Every 4–6 days, half of the medium was removed and replaced with fresh medium until passaging.A healthy live flounder weighing 123.5 g was selected to be anaesthetized by 40 mg mlWhen the cells migrating from the muscle tissue pieces stopped growing, the cell clusters were scattered with trypsin digestion (0.25% trypsin and 0.2% EDTA in PBS) and seeded in the original culture flask again. In order to protect the cells’ proliferation activity, upon attaining 80% monolayer, above trypsin digestion was followed to subculture the cells every 5–7 days at 1:1 cell suspension to fresh medium according to the different growth status.A healthy triploid flounder weighing 131 g was selected and performed on the same day using the similar method of the diploid flounder as mentioned above, but the selected explant (white muscle tissue) was close to the skeleton. In addition, low concentration of trypsin digestion (0.05% trypsin and 0.04% EDTA in PBS) was used in the first passage when the cell clusters were seeded in a new culture flask.S(2n) and POMSCS(3n) were separately suspended in frozen stock solution ) at a density of 1–3×106 cells ml−1. The cell suspension was dispensed into 2 ml cryogenic vial labeled with the cell name, freezing serial number and date, and kept in a NALGEBETM Cryo 1 °C Freezing Container  at 4 °C for 30 min, −20°C for 2 h, −80°C overnight. Then these vials were transferred into liquid nitrogen (−196 °C) for long-term storage. To recover and reseed the cells, the frozen cells were taken out from liquid nitrogen and quickly thawed in 42 °C water bath. Following removal of the freezing medium by centrifugation , cells were transferred into a new flask and resuspended in complete MEM medium, and cultured at 25 °C.For cryopreservation, the cells at passage 10, 20, 30, 40, 50 and 60 from POMSC4 cells ml−1 at passage 48 of POMSCS(2n) and passage 47 of POMSCS(3n) were severally seeded into 24-well plates and incubated at 25 °C. On day 1, 3, 5, 7, 9 and 11, four wells of POMSCS(2n) and POMSCS(3n) cells were respectively counted using a haemocytometer. Cell growth curves were drawn, and the population doubling time (PDT) was calculated according to the equation PDT = t∗[lg2/(lgtN −lgoN)]  . Firstly, 9.0×10 at t h) .S(2n) cells at passage 30 and POMSCS(3n) cells at passage 29 were prepared to analyze chromosomal karyotype. Briefly, 1.0×106 cells were separately inoculated into a 25 cm2 culture flask and incubated at 25 °C overnight. After 24 h, the cells were subsequently incubated at 25 °C with colchicine (1.0 µg ml−1) for 3 h in the same flask, and then the monolayer was trypsinized and harvested by centrifugation . The supernatant was discarded and the cells were suspended in 10 ml hypotonic solution of 0.075 mol L−1 KCl for 25 min at 37 °C, then prefixed 5 min in 2 ml of cold fresh Carnoy’s fixative (methanol: acetic acid = 3:1) by centrifugation . Subsequently, the cell pellets were fixed twice in 5 ml cold Carnoy’s fixative, 15 min for each time. After centrifugation , cells were suspended in 0.5 ml cold Carnoy’s fixative. Glass slides were prepared using the conventional drop-splash technique and air-dried. Chromosomes were stained with 10% Giemsa for 10 min. One-hundred photographed cells at metaphase were counted under an Eclipse 80I fluorescence microscope . The chromosomal karyotypes were analyzed according to POMSCS(2n) and POMSCS(3n) cells were respectively calculated according to the measurement values of 20 cells under the Eclipse 80I fluorescence microscope.In the meantime, the nuclear-cytoplasmic ratios of POMSCpax7b (S(2n) at passage 53 and POMSCS(3n) at passage 52 using RNA isolation kit . The RNAs were incubated with RNase-free DNase I  to eliminate contaminating genomic DNA before being reverse-transcribed into cDNA using oligodT primers and M-MLV reverse transcriptase  according to the manufacturers’ instructions. PCR was carried out in a volume of 25 µl containing 1 µl (400 ng) of cDNA as template, 0.5 µl of each primer (10 µM), 10.5 µl nuclease-free water and 12.5 µl of 2×Pfu MasterMix . PCR was run as follows: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 52 °C for 30 s and 72 °C 30 s, and then 72 °C 10 min for elongation. A RT-PCR minus control was also included. The 198bp PCR products were analyzed by 1% agarose gel electrophoresis.The cell types of the two cell lines were verified with analysis of pax7b  skeletalS(2n) cells at passage 56 and POMSCS(3n) cells at passage 55 were examined for the expression of Desmin as a myogenic cell marker 5 cells were respectively inoculated in one 24-well plate and incubated at 25 °C for 72 h. Cells were washed three times in cold PBS, fixed in paraformaldehyde  for 10 min at room temperature, washed for 5 min in cold PBS, perforated 15 min in cold 0.5% Triton X-100 PBS, washed twice in PBS, blocked for 30 min in 1% BSA, and then incubated with the primary antibody  dissolved in 200 µl 1% PBS) at 4 °C overnight. After washed twice in PBS, cells were incubated with the appropriate secondary antibody (2.5 µl Biotin-labeled Goat anti-Rabbit IgG (H + L) antibodies  dissolved in 200 µl 1% PBS) at 25 °C for 1 h. Then washed in PBS once more, and peroxidase activity was visualized with 3, 3′ N-Diaminobenzidine Tertrahydrochloride  in Tris–HCl buffer (pH 7.6) containing H2O2. Labelled cells were examined under the Eclipse 80I fluorescence microscope. Negative controls (without the primary antibody) were included in the experiment.The POMSCS(2n) cells at passage 32 and POMSCS(3n) cells at passage 33 were individually inoculated into a 12 well plate at a density of 1–2×104 cells/ml in growing medium , 10 ng ml−1 bFGF, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin). After 2 days, differentiation induction of cells was separately performed by switching 80–90% confluent cells to differentiation medium (DM) supplemented with 2% horse serum , 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. The medium was changed once every 2 days. Control groups with GM all along experimental period were performed. Cells from induction and control groups were respectively harvested on the 6th day. The 4′, 6-diamidino-2-phenylindole  was used to stain the cell nucleus. In detail, cells were washed in cold PBS, fixed in paraformaldehyde  for 10 min at room temperature, washed 5 min in cold PBS, perforated 15 min in cold 0.5% Triton X-100 PBS, washed twice in cold PBS, stained with 0.5 mg/ml DAPI for 2 min. Then, labelled cells were visualized under the Eclipse 80I fluorescence microscope. The differentiation experiment had three replicates.POMSCS(2n) and POMSCS(3n) at passage 40 and 39 were respectively inoculated into 12-well plates at a density of 1–1.5×105 cells/ml in growing medium GM. After 48 h, confluent cells were switched into DM. The medium was changed once every 2 days. On the 4th day of differentiation, cells were harvested. Simply, cells were washed in PBS, fixed in paraformaldehyde , perforated in 0.5% Triton X-100 PBS, blocked in 1% BSA, and then incubated with the primary antibody  dissolved in 200 µl 1% PBS) at 4 °C overnight. After washed in PBS, cells were incubated with the secondary antibody (1.5 µl Goat anti-Mouse IgG (H + L) Secondary Antibody, Cy3 conjugate  dissolved in 200 µl 1% PBS) at 25 °C for 1.5 h. Then cells were washed in PBS once more and stained with 0.5 mg/ml DAPI for 2 min. Labelled cells were observed under the Eclipse 80I fluorescence microscope. The differentiation experiment had three replicates. Negative controls (without the primary antibody) were included in the experiment.To confirm that the long fibre-like cells after differentiation are myotubes, cells of POMSCS(2n) cells from passage 24 and POMSCS(3n) cells from passage 22 were respectively seeded in a 24-well plate at a density of 4×105 cells well−1. Sub-confluent monolayers were separately transfected with pEGFP-N3 express vector  using lipofectamineTM 2000 . Briefly, 1 µl lipofectamineTM 2000 and 2 µl pEGFP-N3 (400 ng µl−1) were dissolved respectively in 49 µl and 48 µl of MEM (without FBS or antibiotics). After 5 min, the two solutions were mixed and incubated for 25 min. Subsequently, the mixture was added gently into the well and the cells were cultured for 4 h at 25 °C, then the supernatant was substituted with fresh complete medium immediately. The green fluorescence signals were observed under an Eclipse TE2000-U fluorescence microscope  during the next one week regularly.POMSCS(2n) and POMSCS(3n) were analyzed with the SPSS package for Windows . Significant differences were determined using One-way ANOVA tests followed by Duncan’s multiple comparison tests at the probability level of 0.05. Data from POMSC2 culture flask, cells began to migrate from the edges of the seeded tissue samples. Confluent monolayers of cells were observed on the 8–12th day (S(2n) and POMSCS(3n)) from flounder skeletal muscle were established (S(2n) and POMSCS(3n) cell lines have been subcultured for 67 passages and 66 passages, respectively. Morphologically, the cell lines are both composed of mainly spindle cells and a small number of epithelial cells. The nuclear-cytoplasmic ratios are 0.0409 ± 0.0043 for POMSCS(2n) cells at passage 26 and 0.0546 ± 0.0061 for POMSCS(3n) cells at passage 27.Approximately 4–7 days after the muscle tissue fragments adhered to the bottom of the 25 cm12th day  and 1D. ablished  and 1F. S(2n) and 85–90% for POMSCS(3n). Both cells successfully grew to confluence in 5 days. Almost no apparent morphological differences compared with fresh cells were observed.Cells were cryopreserved at different passages in liquid nitrogen (−196 °C). After storage of 3–5 months, the cells were successively thawed and seeded into flasks with survival rates of 90–95% for POMSCS(2n) and POMSCS(3n) were different from the typical “S” shape, which all showed a sharp rise and drop in the growth tendency (t∗[lg2/(lgtN − lgoN)], the doubling times were 60.92 ± 2.17 h at a inoculum density of 8.70 × 104 cells ml−1 for POMSCS(2n) and 69.88 ± 0.37 h at a inoculum density of 8.19×104 cells ml−1 for POMSCS(3n). It revealed that POMSCS(2n) cells proliferated more rapidly than POMSCS(3n) cells (P < 0.05) when cultured at the same temperature, media and serum concentrations. Both growth curves of POMSCtendency . The plaS(2n) and POMSCS(3n) are 48 and 72 counted at passage 30 or passage 29, respectively. The metaphases both displayed normal karyotype morphology, and their chromosomal karyotypes are 2n = 48t and 3n = 72t. The chromosome assays showed that the chromosomes of cells were all telocentrics . The modpax7b (S(2n) cells at passage 53 and POMSCS(3n) cells at passage 52. Sequencing results showed 100% match with the known flounder gene sequence. The types of these two cell lines should be skeletal muscle satellite cells. The expected 198bp fragment of pax7b  was respS(2n) and POMSCS(3n) were respectively labeled by Desmin antibodies, although the intensity of the labelling varied (S(2n) and POMSCS(3n) should be myogenic cells. The cells of POMSCg varied . It provS(2n) and POMSCS(3n), cells were plated at a low density firstly in GM, then switched to DM. At 48 h after changed to DM, long fibrous cells could be observed in POMSCS(2n) and POMSCS(3n) compared with the controls (S(2n) or POMSCS(3n) but the control groups (S(2n) and POMSCS(3n) cells have the differentiation abilities to differentiate into small multinucleated myosatellite fibres. In order to analyze the differentiation ability of POMSCcontrols . The tral groups  and 7B. S(2n) and POMSCS(3n) cells were first cultivated in GM for 2 days, then switched to DM. On the 4th day of differentiation, anti-myosin heavy chain antibody staining was performed to demonstrate the expression of sarcomeric myosin proteins. Cells of POMSCS(2n) and POMSCS(3n) both displayed positive of myosin heavy chain (MyHC) (POMSCn (MyHC) , which wS(2n) and POMSCS(3n) cells were successfully transfected with pEGFP-N3 reporter gene, and the expression of EGFP in these cells could be detected after 16 h (S(2n) cells and15–25% in POMSCS(3n) cells, respectively. Results indicated the suitability of POMSCS(2n) and POMSCS(3n) for transfection. Both POMSCter 16 h . The numin vitroThe culture condition selection of POMSCs S(2n) and POMSCS(3n) were respectively subcultured for 67 passages and 66 passages. The primary culture protocol of MSCs similar to the mammals was carried out in gilthead sea bream and trout  could stimulate cell proliferation of MSCS . In part in vitro. Accordi tissues .S(2n) and POMSCS(3n) both showed a steep rise and down, which were different from the typical “S” shape. The specific reason is not clear at present. Differentiation experiment proved that the cells were capable of differentiating into the multinucleated cells in vitro which was the basis of myotube and muscle fibre. The myosatellite cells become increasingly diluted with the age of fishes, but as the crucial basis of the muscle hyperplasic and hypertrophic growth, the total number of myosatellite cells in the white axial muscle might be rather constant during the growth phase of the fish (S(2n) and POMSCS(3n). And the cells of POMSCS(2n) and POMSCS(3n) after differentiation displayed positive to MyHC, which was considered as the differentiation index  cells was 1.33 times of POMSCS(2n) as expected. The proliferation rate of POMSCS(3n) was slightly slower than POMSCS(2n). While POMSCS(3n) had stronger differentiation capacity than POMSCS(2n)s. In the differentiation experiment, more multinucleated cells, especially myotubes with 3–4 nuclei, could be observed in POMSCS(3n) than POMSCS(2n).Our results exhibited different characters between triploid and diploid myosatellite cells. The nuclear-cytoplasmic ratio of POMSCS, on activation, participate in myogenic function and provide the biological basis for longitudinal and cross-sectional growth  showed a higher proliferation rate than POMSCS(3n), and POMSCS(3n) had a stronger differentiation capacity than POMSCS(2n). These results could provide a new understanding on the cytological mechanism of muscle growth and development in diploid and polyploid fish species in flounder and other marine fishes. The two cell lines will become the useful tools in the researches concerning the gene function analysis, environment toxicology, pharmacology and so on.Two myosatellite cell lines 10.7717/peerj.1519/supp-1Supplemental Information 1The resulting of the cell counting that drawing the growth curve of cells.Click here for additional data file.


Dear Editor,Intracellular trafficking is a basis of cellular activities, including cell migration, immune response, and development  to humans. In the C.elegans intestine, Rab10 coordinated the transport of clathrin-independent cargo between early endosomes and recycling endosomes   were homozygous mutants and 1 (2.9%) was a resorbed embryo; out of 71 E8.5 embryos, 8 (11.3%) were homozygous mutants and 10 (14.1%) were resorbed embryos , 10.5, and 9.5 were collected and genotyped by PCR. As shown in Fig. Rab10−/− embryos was due to the failure of cell proliferation. The embryos were separated at E7.5 after fertilization to obtain 10 μm frozen sections for immunofluorescence. As shown in Fig. Based on our observation of E7.5 developmental failure above, we used an immunofluorescent assay with Ki67 (Abcam) and 4’,6-diamidino-2-phenylindole (DAPI) staining to determine whether defective development of Rab10−/− embryos  staining was used to detect the ratio of apoptotic cells in wild-type and yos Fig. B. EmbryoRab10−/− mutants was reduced by 13% compared with that of the wild-type embryos. The diameter of the nuclei in wild-type embryos was 11.9 μm, whereas in Rab10-null mice, the diameter was 10.3 μm  because the large scale of the nucleus occupied most of the cytoplasmic space and the resolution was limited. We then used transmission electron microscopy to study the ultrastructure in Rab10−/− samples. As shown in Fig. Rab10−/− cells, many vacuoles were accumulated in the cytoplasm , tissue-specific knockout mouse models are needed to further define its physiological functions at different tissues.This work is the first attempt to access the physiological function of Rab10 protein using a


Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P.falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described.Semi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Nicotiana benthamiana in the context of human full-size IgG1:κ. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A.B lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in −7 M [humAb10.1 (H5F6:κ5E8)], 5.46 × 10−9 M [humAb10.2 (H5F6:κ5F6)] and 4.34 × 10−9 M [humAb10.3 (H5E8:κ5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6–6.6 mg/ml], 6.9 mg/ml (CI 5.5–8.6 mg/ml) and 9.5 mg/ml (CI 5.5–16.4 mg/ml), respectively.Supernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10P. falciparum MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.This report describes a platform for the isolation of human antibodies from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs directed against  Malaria still claims over half a million victims each year and poses a significant burden to global health care and to the economy of endemic countries . In humaPlasmodiumfalciparum encodes more than one MSP that contains a double epidermal growth factor (EGF)-like domain, Black et al. sought to identify potential homologs of MSP1. It appears that MSP1 and MSP10 have several features in common; i.e. they share a double EGF-like domain and a glycosylphosphatidylinositol (GPI) anchor at their C-terminus and both are mainly expressed during the later blood stages. Furthermore, MSP10 is also subject to proteolytic processing similar to MSP1, i.e. in the case of MSP10 starting from 30 h post invasion a product of 36 kDa can be detected besides the intact protein of 80 kDa. Puentes et al. aimed at finding out if certain parts of MSP10 are capable of binding to erythrocytes and to inhibit the invasion of merozoites into new erythrocytes . Erythrocytes of 12–16 individual donors, all having the blood group 0, Rh-positive, were obtained in CPDA tubes from the Department for Transfusion Medicine, RWTH Aachen University Clinic, Aachen, Germany. The suspensions were mixed, centrifuged in order to remove plasma and white blood cells  and washed for three times in SAG-mannitol -mannitol). Washed erythrocytes were taken up in SAG-mannitol to obtain a final haematocrit of 66.6% (v/v).d before . BrieflyP.falciparum as described before using humAbs in final concentrations ranging from 10 to 0.078 mg/ml in RPMI 1640 containing 25 mM HEPES /[A655 nm (schizont growth control) − A655 nm (erythrocyte control)].In vitro growth inhibition of the isolated antibodies was assessed via GIA on mM HEPES . As posimM HEPES . As corrmM HEPES . P.falci the GIA . Schizon the GIA . The perP.falciparum 3D7A were used to prepare IFA slides. Smeared parasites were fixed in methanol at −20°C for 10 min. After blocking in 1% (v/v) FCS (low IgG) in PBS for 3 h at 25°C, slides were incubated with 50 µg/ml mouse-anti-MSP4 and either of the three human-anti-MSP10 antibodies (10 or 25 µg/ml) or 2G12 (an anti-gp 120 HIV-1 humAb as isotype control for human IgG) in 1% (v/v) FCS (low IgG), 0.01% (w/v) Saponin in PBS) in a moist box at 25°C for 1 h. After four washing steps in PBS, the slides were treated with 1:100 goat-anti-mouse IgG(H + L)-Alexa Fluor® 488 (Life Technologies), 1:1,000 goat-anti-human IgG(H + L)-Cy3 (Dianova) and 10 µg/ml Hoechst 33342 (Life Technologies) in a moist box at 25°C for 1 h. Five washing steps were carried out. Prolong® Antifade mounting medium (Life technologies) was used to conserve the staining. Images were taken at a 630× magnification with a Leica TCS SP8 microscope and LAS AF Version 3.1.3 software .Synchronous Kinetic binding parameters of the monoclonal antibodies were analysed by SPR spectroscopy using a Biacore T200 instrument  and Protein A-functionalized CM5-S series sensor chips as previously described . For kinThe study was designed to efficiently isolate humAbs which recognize the EGF-like domains of MSP10 and which represent a part of the naturally-acquired, protective immunoglobulin repertoire of semi-immune donors. The definition of semi-immunity of the plasma donors was primarily based on observation that they had not shown any signs of clinical malaria for 2 years. Moreover, several screenings had been performed to make sure that the set of donors had a broad anti-malarial antibody response, as published before , 37–39.To quantify the MSP10-response, plasma of 31 Ghanaian blood donors were screened for reactivity against DsRed-MSP10(EGF1) Figure a and DsRIt had been reported that naturally acquired antibodies which are specific for plasmodial EGF-like domains primarily react with conformational epitopes , 40, 41.+, merozoite surface antigen-specific B cells as basis for the subsequent EBV transformation, PBMCs from donor G13, one of the donors whose plasma reacted with both of the MSP10 EGF-like domains, were used. An amount of 1.25 × 107 vital PBMCs was subjected to flow cytometric sorting and 4,869 vital CD22+/IgG+ B cells (0.039%) were selected and seeded for EBV transformation. After 7 weeks, 6 out of 90 wells (6.67%) showed reactivity with the multi-domain fusion protein E25T, which includes both EGF-like domains of MSP10. The corresponding LCLs were expanded. Fifteen weeks after EBV transformation four of these six cultures had ceased to secrete specific IgG. The remaining two cultures, 5E8 and 5F6, were used for clonal analyses by spectratyping and rescue of Vh and Vl sequences by PCR.For selection of mature, IgGSpectratyping revealed that LCL 5F6 was monoclonal as early as 16 weeks post infection. At that time point LCL 5E8 was still octaclonal. After continuous subculturing of the LCL 5E8 for another 21 weeks, it also became monoclonal.Each of the LCLs 5E8 and 5F6 were subjected to RNA isolation, cDNA synthesis and amplification of immunoglobulin Vh-, Vk- and Vl- region sequences. From each culture, one Vh and one Vk were recovered. Subsequently, the Vh and Vl regions were sequenced and compared to the closest corresponding germline sequences. As shown in Figure Nicotiana benthamiana. Since antibody heavy and light chains are encoded on separate pTRAkt plant expression plasmids it is possible to realize any HC/LC combination. After initial small scale expression studies three  of the four possible Vh/Vl combinations recognized MSP10. Subsequently, the three corresponding binding-competent humAbs were transiently expressed at a medium scale (10-16 Nicotiana benthamiana plants). Heavy chain 5F6 (H5F6) was either co-expressed with the light chain 5E8 (κ5E8) or 5F6 (κ5F6), respectively, resulting in humAb10.1 (H5F6:κ5E8) and humAb10.2 (H5F6:κ5F6). Heavy chain 5E8 (H5E8) was co-expressed with its natural light chain (κ5E8), thus forming humAb10.3 (H5E8:κ5E8). The average yield of the recombinant antibodies was 100-200 mg per kg of fresh leaf material.Variable region sequences of 5E8 and 5F6 were cloned into pTRAkt vectors for recombinant transient expression in The reactivity of the humAbs against MSP10(EGF1) and MSP10(EGF2) was verified by ELISA with DsRed-MSP10(EGF1), DsRed-MSP10(EGF2) and multi-EGF comprising both EGF-like domains of MSP10. All humAbs bound to multi-EGF Figure . The reca, kd and KD of both humAb10.2 and humAb10.3, produced in different expression systems, were determined. The KD values of all antibodies are summarized in Table a) for both humAb10.2 and humAb10.3 were observed. However, the dissociation rate (kd) of humAb10.3 was lower than the one of humAb10.2, indicating that humAb10.3 possesses a higher affinity for MSP10(EGF2) than humAb10.2 for MSP10(EGF1). The affinity of the humAb10.1, which is composed of the artificial combination of H5F6:κ5E8, to its antigen MSP10(EGF1) was more than 100-fold lower than the affinity of humAb10.2 for the same antigen. The recombinant humAb10.2 and the corresponding LCL-derived 5F6 antibody showed a comparable affinity to the antigen MSP10(EGF1). Surprisingly, the affinity of the humAb10.3 expressed in plants differs by a factor of seven from the respective antibody produced by the corresponding LCL (5E8).The affinity of the humAbs to MSP10 was analysed by SPR spectroscopy using the multi-EGF fusion protein, which is monovalent and contains several EGF-like domains of the MSP family including both EGF-like domains of MSP10. Kinetic constants kYY) and one residue in the CDR3 (DSAVYYCQQYED) of the heavy chain of humAb10.3 were predicted to be sulfated in mammalian expression systems.A potential explanation for this difference might be a posttranslational modification of the antibody, e.g. the sulfation of tyrosine residues, which is assumed not to take place in plants . TherefoKnowing that antibodies of the protective immunoglobulin repertoire of semi-immune individuals mainly recognize correctly folded plasmodial EGF-like domains as shown in Figure In conclusion, all human antibodies described here only bind the native antigen, thus recognizing a conformational epitope. Secondly, all three antibodies bind highly specific to MSP10 EGF-like domains, and do not show any cross-reactivity Figure .Figure 6P.falciparum 3D7A schizonts were used for confocal microscopy with humAb10.1, humAb10.2 and humAb10.3  2.6–6.6 mg/ml), humAb10.2 inhibited the parasite growth with an EC50 value of 6.9 mg/ml (CI 5.5–8.6 mg/ml) and humAb10.3 inhibited the merozoite invasion with at an EC50 value of 9.5 mg/ml (CI 5.5–16.4 mg/ml).In order to test the inhibitory potential of humAb10.1, humAb10.2 and humAb10.3, an in vitro-GIA with A Figure . HoweverPlasmodium vivax DBP [3-repeat of CSP) [So far, only a handful of anti-plasmodial humAbs have been isolated and described. Some of these are specific for the MSPs  or GLURP –46. Otheivax DBP –49. Anot of CSP) , 51.Here, additional human antibodies specific for the merozoite surface, isolated by an innovative human antibody technology platform, are presented. This technology combines the screening of naturally exposed semi-immune individuals from endemic countries for antigen-specific reactivity, the targeted selection of antigen-specific B cells by flow cytometric sorting, the subsequent EBV transformation, rescue of the antibody V region sequences and subsequent recombinant production in plants. This allows to produce different versions of the antibody, the antibodies produced directly in the LCLs, as well as the recombinant antibody. Combinations of different heavy and light chains can only be produced using recombinant antibodies. Moreover, the slight difference in affinity of humAb10.3, which likely relies on posttranslational modifications, can only be detected when comparing multiple expression systems.The rescue of the antibody variable sequences is especially important, as LCLs tend to cease to secrete antibodies or stop to proliferate altogether, which would lead to an irreversible loss of the desired immunoglobulin . In contNicotiana benthamiana are correctly folded. The general reactivity of these plasma samples against various P.falciparum antigens has previously been shown elsewhere [19 [The first step of the procedure, the screening of plasma for specific antigen-binding, provides valuable information: On the one hand, the positive reactivity shows that specific antibodies are present in the subjects tested; on the other hand, it validates that the recombinant antigens produced in lsewhere , 37–39. here [19 . Moreovehere [19 . CoherenIt was found that each of the isolated antibody chains had accumulated a considerable number of coding mutations during their affinity maturation in vivo. It can be assumed that it takes 4 years to accumulate 20 mutations in the Vl and 30 mutations in the Vh sequences for a desirable affinity maturation to occur . ConsequBy ELISA and SPR spectroscopic measurements it was demonstrated that 1) the correct variable regions of LCLs 5E8 and 5F6 were rescued and that (2) the corresponding recombinant IgG1:κ full-size antibodies shared the same characteristics with their naturally produced counterparts. It appears that the correct variable sequences had been recovered, cloned and expressed in the context of IgG1:κ given the facts that (1) LCLs 5E8 and 5F6 were monoclonal by the time of the rescue of their variable immunoglobulin regions and that (2) naturally and recombinantly produced humAb10.2 and humAb10.3 showed the same respective specificities. Interestingly, the artificial combination H5F6:κ5E8 of humAb10.1 featured the same specificity for MSP10(EGF1) as humAb10.2 (H5F6:κ5F6). However, humAb10.1 was much less affine for MSP10(EGF1) than humAb10.2  depending on their activity [Puentes et al. found three synthetic 20-mer peptides of MSP10 to be binding to human erythrocytes and to be hampering the invasion of merozoites into erythrocytes in vitro. Interestingly, one of these peptides corresponds to a part of the first EGF-like domain of MSP10 and showed 65% (±2%) inhibition at a concentration of 200 µM (0.46 mg/ml) . The EC5activity . Given tHowever, besides mere binding to the target, antibodies may also fulfil their function in vivo by initiating the complement cascade  and/or b19-specific antibodies may compromise the intraerythrocytic development of Plasmodia [Besides a function during attachment to/invasion of new erythrocytes, the EGF-like domains of MSP10 might be carried into the erythrocyte during invasion and might have a function during the development of merozoites into rings. EGF-like domains of MSP1, MSP2 and MSP4 have been reported to stay attached to the merozoite membrane by GPI anchors after processing and to be carried into the freshly infected erythrocyte , 65, 66.lasmodia , 68. Thulasmodia .The presented work describes the generation and characterization of three humAbs which are specific for the first or the second EGF-like domain of MSP10, respectively. To the best of our knowledge this study is the first to combine flow cytometric selection of antigen-specific B cells with EBV transformation and to successfully generate humAbs by this very technique. To our knowledge, these are the first human monoclonal antibodies directed at MSP10, which have been isolated. HumAb10.2 and humAb10.3 possess the genuine combination of corresponding heavy and light chains, respectively; they are highly specific for their corresponding plasmodial EGF-like domain and bind these with high affinity. In in vitro GIAs, the MSP10-specific humAbs demonstrated significant inhibition of the parasite strain 3D7A even though comparatively high antibody concentrations were required. Further experiments should address (1), the potential role of immune effector cells in inhibition of merozoites by MSP10-specific antibodies and (2), the identification of interaction partners of MSP10 to deepen understanding of its function(s). The generated humAbs represent a valuable tool to tackle these MSP10 related questions in the future.


HIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-κB and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1.Here we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9’s Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1.We identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs. Despite efficient antiretroviral therapy, eradication of human immunodeficiency virus (HIV) 1 infection is challenging and requires novel biological insights and therapeutic strategies. Eradication of latent HIV-1 provirus is especially challenging as integrated HIV-1 is not affected by the existing anti-HIV-1 drugs unless viral transcription is activated . HIV-1 TThus, studies from our group and others showed that PP1 is an important regulator of HIV-1 transcription. We recently developed a panel of small molecular compounds targeted to a non-catalytic site of PP1 and identified 1H4 compound that efficiently inhibited HIV-1 transcription and replication . We furtWe recently developed a small molecule, 1E7-03, that targets a non-catalytic site of PP1, interfered with the binding of Tat to PP1 and inhibited HIV-1 transcription and replication . The 1E7env, gag and nef RNA expression in PBMCs obtained from both donors as detected by quantitative RT-PCR . PBMCs obtained from two different donors, a Caucasian and an African American, were infected with HIV-1 Luc and treated with the compounds 1–4. At 10 μM concentrations, SMAPP1 showed the best activating capability in a Caucasian donor-derived cells Figure a, b, wheR Figure c, d, sugAt the same time, this compound showed no significant effect on cell viability as detected by the trypan blue exclusion assay Figure e. Taken To test the effect of SMAPP1 on transcription of the integrated HIV-1 genome, we analyzed HIV-1 RNA production in chronically infected T lymphocytes (ACH-2) treated with this compound. As a positive control, we used a potent inhibitor of histone deacetylases (HDAC), suberoylanilide hydroxamic acid (SAHA)  which waTo test the effect of PP1 activation on HIV-1 transcription in the model of latently infected primary cells, the PBMCs isolated from healthy donors were infected with HIV-1 isolate NL4-3, treated with IL-7, cultured for 15 days to transfer T cells to the quiescent stage , and theThus, our data indicates that, SMAPP1 moderately activated HIV-1 NL4-3 transcription in chronically infected T cell lines and low-productively infected quiescent T cells. Despite the lower impact of SMAPP1 on HIV-1 transcriptional activation than the HDAC inhibitor SAHA and PKC agonist bryostatin 1, the tested PP1 targeting agent had no visible cytotoxicity at effective concentrations. Activation effect on the latent infection caused by HIV-1 subtype C isolate has not been observed.E. coli cells  of CDK9 as CDK2 phosphorylation site and showed that its phosphorylation induces HIV-1 transcription (reviewed in ). We alsTo determine whether SMAPP1 has a global effect on protein expression in T cells, we conducted proteomic analysis of SMAPP1-treated CEM T cells using a recently developed protocol in which tryptic peptides from total cell lysates were fractionated on a cation exchange column . In totaTo further analyze the effect of SMAPP1 on PP1, we conducted an in silico docking analysis of the molecule on the surface of PP1 using ICM software and coordinates of the PP1 crystal structure. Molecular model of PP1α was built with ICM-Pro software package using PDB structure 3E7A . SMAPP1 Taken together, our results indicated that we have identified a small molecule compound, SMAPP1, which can induce HIV-1 transcription and reactivate latent HIV-1 provirus. This compound increases CDK9 phosphorylation on several key residues and activates PP1 in vitro and also induces Sds22 expression in the treated T cells.In the present study, we extended our previous findings of the small molecule mimetics of the “RVxF”-docking peptide by identifying compounds that activate HIV-1. We previously identified HIV-1 inhibitory 1H4 compound  and, morWe used several cell-based models of HIV-1 latency to test the effect of PP1-targeting small molecule compounds on the activation of viral expression. These include one round HIV-1 infection, latently infected Jurkat (lymphoblastoid T cells), THP (monocytes) cell lines, chronically infected CEM T cells (ACH-2) and primary CD4+ T cells within the pool of infected PBMCs treated with IL-7 to transfer them to quiescent phase. While SMAPP1 induced HIV-1 in chronically and latently infected T cell line two to threefold, in PBMCs we could not detect significant effect on total HIV-1 transcription, whereas a trend to transcription reactivation, especially in the case of HIV-1 subtype B infection, was observed. On the other hand, analysis of HIV-1 gene expression in acutely-infected PBMCs from various donors, treated with SMAPP1, showed increased transcription of HIV-1 mRNA; the level of both unspliced and single-spliced mRNA species was detected. High variability of SMAPP1 effect on HIV-1 transcription in the cells from different donors, as well as lack of visible effect on the transcription of HIV-1 subtype C virus genome within the low-productive infection in primary cells suggests that impact of this compound on HIV-1 infection depends on the nature of infected cells and probably the virus subtype.Our previous studies showed that HIV-1 Tat interacts with PP1 and translocates it to the nucleus . PP1-tarWe analyzed the effect of SMAPP1 on PP1 in vitro, using a Biacore assay with Chip-immobilized bacterially expressed PP1. While we observed relatively weak binding with Kd = 183 μM, the presence of SMAPP1 induced dephosphorylation of a peptide substrate containing an RVxF motif suggested that it may interfere with the binding of RVxF motif or some other sites where the hybrid substrate interacts with PP1. The latter is further supported by the in silico docking analysis, which showed preferred interaction of SMAPP1 with the C-terminal groove of PP1, which may also be involved in the interaction of the peptide substrate with PP1. Analysis of proteins expressed in T cell treated with SMAPP1 showed upregulation of the PP1-regulatory subunit, Sds22. Expression of Sds22 was also verified by immunoblotting analysis. Sds22 is evolutionarily conserved ancient interactor of PP1 that along with Inhibitor 3 forms a complex with PP1 and helps to translocate it to the nucleus . Sds22 aOur study shows that HIV-1 can be induced by a small molecular compound that interferes with PP1 . The cAR−E− , as well as latently-infected CEM subclone ACH-2 2+, 3.0 for [M + 3H]3+ and higher charge state were considered. These SEQUEST criteria thresholds typically result in a 1–2% of False Discovery Rate. FDR was determined by searching on a decoy database. We used SIEVE 2.1 software (Thermo) for label-free quantitative analysis of the high resolution MS spectra produced by Orbitrap MS scans. We also explored protein networks in SMAPP1-treated cells by uploading the results of SIEVE 2.1 analysis into Ingenuity (Qiagen) and performing pathway analysis.E. coli cells (Invitrogen) were co-transformed with al vector RP1B, which expresses human PP1α (residues 7-300), and pGR07, which expresses GroEL/GroED chaperones . The cells were grown in media supplemented with 1 mM MnCl2 at 30°C to an A600 ~ 0.5. Then arabinose (2 g/L) was added to induce expression of the GroEL/GroES chaperones. When A600 ~ 1 was reached, the cells were transferred to 10°C and PP1 expression was induced with 0.1 mM IPTG for 20 h. Harvested cells were lysed using sonication in a solution containing in 50 mM Tris–HCl (pH 8.0), 5 mM imidazole, 700 mM NaCl, 1 mM MnCl2, 0.1% Triton X-100 (v/v) and protease inhibitors. His-tagged PP1 was purified using a Ni–NTA IMAC column (Qiagen). PP1 was then dialyzed and stored at −70°C in 50 mM Tris–HCl (pH 8.0), 5 mM imidazole, 700 mM NaCl, and 1 mM MnCl2. PP1 activity was then assayed as previously described [BL21 (DE3) 2) and loaded with 0.5 mM NiCl2 at a flow rate of 10 μL/min for 180 s. After NiCl2 injection, both flow cells were washed twice with the running buffer at flow rate of 100 μL/min for 40 s. PP1 was diluted with the running buffer to a concentration of 200 nM and was then passed over the second flow cell of the sensor chip at a flow rate of 5 μL/min. The final amount of PP1 immobilized on the surface was 3,500 RU. The first flow cell was only loaded with Ni2+ and was used as a reference for non-specific background binding during the experiment. For binding and kinetics experiments, all compounds were diluted in the running buffer, at 50, 25, 12.5, 6.25, 3.125, 1.56 and 0 μM and were passed over the two flow cells at a flow rate of 100 μL/min for 60 s. The number of response units was recorded after the subtraction of the reference flow cell’s value (Fc2-1). Three repetitions were performed for each injection. Data were analyzed using the BiaEvaluation software of Biacore with a 1:1 binding model.All SPR measurements were conducted on a Biacore T200 instrument  at 25°C. Recombinant His-tagged PP1 was immobilized on a Ni–NTA sensor chip . The two flow cells of the sensor chip were primed with running buffer . 3D atomic structures of PP1α were taken from the PDB database . Preliminary analysis showed high similarity of these structures (mean pairwise RMSD of Cα atoms of the main protein chain was below 0.5 Å). Structure 3E7A  with the±SEM. Differences between any two groups were compared with the unpaired two-tailed Student’s t test on GraphPad Prizm 4.01 software .Results are expressed as mean ± SD or


In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L−1 (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix , glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy.This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 μmol·L It infects around 90% of the world’s population and may be asymptomatic during the life of the host. Its latent state could be regarded as the causative agent of infectious mononucleosis and has been associated with several malignancies including Burkitt’s lymphoma, oral and gastric carcinomas , Hodgkin’s disease, lymphoproliferative disorders in immunodeficient individuals, such as post-transplant lymphoproliferative diseases and a subset of T and NK cell lymphomas –5.EBV was discovered 40 years ago from examining electron micrographs of cells cultured from Burkitt’s lymphoma that indicated a viral etiology and became the first of an unexpectedly wide range of associations discovered between this virus and tumors . It is cin situ hybridization, immunohistochemistry and electron microscopic immunocytochemistry. These techniques have a high cost, demanding skilled labor and time-consuming analysis 4−/[Fe(CN)6]3− or Ru(NH3)62+ (The modified graphite electrodes were studied in aqueous solution containing [Fe(CN)(NH3)62+ .6]4−/[Fe(CN)6]3−, the current of the modified electrode decreases, as well as a larger separation of the peak potentials is observed, denoting difficulty and, eventually, hindrance of the electron transfer to the [Fe(CN)6]4−/[Fe(CN)6]3− system. Such behavior is also seen in other polymer layers , 13.2 ± 3.5 nm [modified electrode with poly(4-ATP)/EBV1] and 31.8 ± 5.2 nm [modified electrode with poly(4-ATP)/EBV1:EBV2].As shown in 3.3.1.−1) was prepared in ethyl alcohol (PA) and H2SO4 solution (0.5 mol·L−1) immediately before use. Phosphate buffer solution 0.1 mol·L−1 was prepared at pH 7.4. All experiments were conducted at room temperature (25 ± 1 °C). The oligonucleotides were synthesized by Invitrogen Life Technologies  with the following sequences: probe: (EBV1): 5′-AGGGATGCCTGGACACAAGA-3′, complementary target (EBV2): 5′-TCTTGTGTCCAGGCATCCCT-3′, non-complementary target: 5′-ACAACCCGTTGG ACTAAC-3′ and (EBV2Mis1): TCTTGTCTCCAGGCATCCCT-3′. Stock solutions of the 3.15 × 10−4 mmol·L−1 probe and 9.45 × 10−4 mmol·L−1 target oligonucleotides were prepared in SSC 6× buffer  and stored in a freezer until use. Buffer components (CH3COOH and CH3COONa or Na2HPO4 and NaH2PO4) were purchased from Sigma-Aldrich Chemical  (ACS purity) and prepared at pH 4.5 or 7.45, respectively. All reagents were used as received. The experiments were conducted at room temperature (25 ± 1 °C).All reagents used were of analytical grade and used without further purification. Ultra-high purity water  was used for the preparation of aqueous solutions. 4-Aminothiophenol  (15 mmol·Lhttp://www.ncbi.nlm.nih.gov/blast).Analysis of the nucleotide sequence  was perf3.2.Electrochemical polymerization and voltammetric measurements were performed using a potentiostat , with a graphite disk (6 mm diameter) cut from a graphite rod  as working electrode. Platinum was used as counter electrode. All potentials are referred to the silver-silver chloride reference electrode (Ag/AgCl). The graphite surface, prior to electropolymerization, was mechanically polished with alumina slurry (0.3 μm diameter), ultrasonicated, washed with distilled water and dried in the air. All solutions were degassed by nitrogen bubbling. Film morphology in absence or presence of biomolecules was assessed through atomic force microscopy (AFM) .3.3.2 prior to electropolymerization. Poly(4-aminothiophenol) films were electrodeposited onto the graphite electrodes from a solution containing 4-aminothiophenol (15 mmol·L−1). The electrochemical experiments were conducted at room temperature (25 ± 1 °C), 50 mV·s−1, −0.4 and +1.0 V, 100 scans in three-compartment cell.The monomer solutions were degassed with N3.4.4Fe(CN)6/K3Fe(CN)6 solution (5.0 mmol·L−1) containing KCl 0.10 mol·L−1 (negative probe) or Ru(NH3)6Cl2 solution (5.0 mmol·L−1) containing KCl 0.10 mol·L−1 (positive probe).The evaluation of ion transport was carried out in K3.5.−4 mmol·L−1 of the probe (EBV1) on the modified electrode surface and dried at the temperature of 37 ± 1 °C. Then the electrode was immersed in 10 mL phosphate buffer with agitation  and dried in N2. The blocking of the binding of non-specific biomolecules on electrode surface was done with BSA 0.5% (w/v) for 3 h, then the electrode was immersed in 10 mL phosphate buffer with agitation  and dried in N2. After that, differential pulse voltammetry (DPV) measurements in one-compartment electrochemical cell connected to a potentiostat were obtained by using 1 mL phosphate buffer  as electrolyte to evaluate the electrochemical sign of modified electrode with the probe.The immobilization of oligonucleotide was carried out by applying two consecutive layers of 13 μL of 3.15 × 103.6.−1) were applied to the modified electrode with EBV1, 126 μmol·L−1. The hybridization was carried out at 57 °C for 20 min. Then the electrode was immersed in phosphate buffer  and dried in N2. The annealing temperature of the oligonucleotide (EBV1) was obtained by gene runner software .For the direct detection, 26 μL of target  were applied on modified electrode. Hybridization was carried out at 57 °C for 20 min. Then the electrode was immersed in phosphate buffer  and dried in N2. In sequence, 18 μL of 1 μmol·L−1 ethidium bromide solution  from Merck Millipore  in ultra-high purity water were applied on the electrode surface during 5 min. Ethidium bromide binds nucleic acids via intercalative mode and causes major changes to DNA. To evaluate electrochemical sign of ethidium bromide, differential pulse voltammetry measurements were conducted using phosphate buffer  as electrolyte for the evaluation of electrochemical sign of electrode modified containing EBV1:EBV2.For the indirect detection, after the immobilization of the oligonucleotide probe (EBV1), 26 μL of target  or one-base mismatch  were added on surface of the sensor subjected to the same process of the complementary target. The hybridization was carried out at 57 °C for 20 min. Detection of complementary target was done indirectly through the use of a mediator.DNA hybridization is based on the ability of the probe to recognize its corresponding complementary target. To verify the specificity of the probe, 26 μL of the non-complementary target . All experiments were conducted at room temperature (25 ± 1 °C).For this study, we added the complementary target concentration normally found in the blood to the solution of the interfering compounds  were stored at 4 °C, protected from light and oxygen during 120 days.3.10.−1 were added to the genosensor. Hybridization was carried out at 57 °C, for 20 min. For the detection, ethidium bromide  was added to the electrode surface, for 5 min.To evaluate the sensitivity of genosensor, 26 μL of different concentrations of complementary target (EBV2), 0, 3.78, 37.8, 378 and 756 μmol·L4.The results showed that functionalized surfaces with poly(4-ATP) are interesting platforms for the development of a genosensor. The produced genosensor shows interesting properties, such as good stability, selectivity and sensibility. This is a promising technique of molecular analysis of a specific biomarker for Epstein-Barr virus. Further studies will extend the system to determination of EBV in serum, plasma, and saliva samples.


Cilia are small antenna-like cellular protrusions critical for many developmental signaling pathways. The ciliary protein Arl3 has been shown to act as a specific release factor for myristoylated and farnesylated ciliary cargo molecules by binding to the effectors Unc119 and PDE6δ. Here we describe a newly identified Arl3 binding partner, CCDC104/CFAP36. Biochemical and structural analyses reveal that the protein contains a BART-like domain and is called BARTL1. It recognizes an LLxILxxL motif at the N-terminal amphipathic helix of Arl3, which is crucial for the interaction with the BART-like domain but also for the ciliary localization of Arl3 itself. These results seem to suggest a ciliary role of BARTL1, and possibly link it to the Arl3 transport network. We thus speculate on a regulatory mechanism whereby BARTL1 aids the presentation of active Arl3 to its GTPase-activating protein RP2 or hinders Arl3 membrane binding in the area of the transition zone.  •Identification of a BART-like protein, CCDC104/BARTL1, as Arl3 binding partner•BARTL1 is a ciliary protein with transition zone localization•Complex structure shows BARTL1 binding to conserved LLxILxxL N-term motif of Arl3•N-terminal amphipathic helix of Arl3 is crucial for its ciliary localization Lokaj et al. biochemically and structurally characterize BARTL1 as an Arl3 effector and speculate on its function in cilia. Cilia are small, microtubule-based antennae-like protrusions of cells critical for the maintenance of cellular homeostasis and many developmental signaling pathways . Small GArl2 and Arl3 (Arf-like) are guanosine triphosphate (GTP)-binding proteins of the Arf subfamily of the Ras superfamily. They switch between an inactive guanosine diphosphate (GDP)-bound form and an active GTP-bound form . This moArl2 and Arl3 are homologous proteins with approximately 52% sequence identity (68% similarity) and very similar structure. In addition, numerous effectors have been identified which interact with the GTP-bound form of both proteins. These are the delta subunit of the photoreceptor-specific phosphodiesterase 6 (PDE6δ) , HRG4/UnDespite the homology in structure and biochemistry, Arl2 and Arl3 may have entirely different biological functions. It was shown very early that transfection of GTPase-negative versions (Q→L) of Arl2/3 and the knockdown by RNAi differentially affect microtubule-dependent processes . Arl2 haArl3-deficient mice revealed that Arl3 is indeed involved in ciliary function affecting kidney and photoreceptor development . BART has been found to be an Arl2-interacting protein , which iD129N containing a C-terminal Strep-flag tag. Such a mutant is expected to associate with GEFs and effectors, as we have shown previously in the identification of plant-specific RopGEFs (Q71L (In a search for ciliary regulators (guanine nucleotide exchange factors [GEFs] and GTPase-activating proteins) for Arl3, we carried out tandem-affinity purifications (TAPs) from HEK293T cells that were transfected with constructs coding for the fast cycling mutant Arl3 RopGEFs . We repe RopGEFs . AlthougFs  cells. In agreement with the literature , we conf133 is not found in cilia  with the truncated protein  were determined by titrating 1 μM Arl3 bound to mant-GppNHp with increasing amounts of effector and measuring fluorescence polarization. Complex formation increases the fluorescence polarization signal and shows that Arl3 binds to BARTL1 or BARTL1133 with KD of 1 or 0.43 μM, respectively -labeled BARTL1. The koff for the complex is 1.7 s−1, which would give an association rate constant of 4 × 106 M−1 s−1 for the interaction between Arl3 and BARTL1, within the normal range for a protein-protein interaction . The asymmetric unit contained two Arl3∙GppNHp and two BARTL1133 molecules . Part of the BART structure in the Arl2∙GTP∙BART complex (PDB: 3DOE)  from α4′ forms a hydrogen bond with the backbone oxygen of Lys9A   was not le to α4 A. The si133 in space group P21, which was solved at 2.0 Å resolution  of 0.788 Ǻresidues , left, wresidues , right. residues C, upper,133 with Arl3ΔN in its active GppNHp-bound state , we set out to speculate on the role of this interaction. We show here that both Arl3 and BARTL1 seem to be ciliary proteins with a partly overlapping localization. While Arl3 is co-staining with acetylated α-tubulin over the entire length of the cilia axoneme and seems concentrated at the transition zone, BARTL1 co-localizes with Arl3 distal to the basal body, corresponding to the transition zone, localized between basal body and cilia axoneme, as shown by co-staining with γ-tubulin as a basal body marker. Staining of endogenous Arl3 in a stable cell line expressing BARTL1-GFP confirms that both proteins are present in the cilia axoneme and around the transition zone . In addi133 to Arl3 reduces the association of Arl3-GppNHp with liposomes.We would like to propose two possible, though not necessarily mutually exclusive, roles for the Arl3-BARTL1 interaction. It has been shown by us and others that the GTP-bound form of Arl3 releases farnesylated and myristoylated ciliary cargo from the transport factors PDE6δ and Unc119a/b. Since this is required for cargo to be transported into cilia, Arl3 is most likely localized as Arl3∙GTP inside cilia. The exclusive localization of active Arl3 inside cilia is guaranteed by the Arl3-specific GTPase-activating protein RP2, which we find enriched around the basal body in IMCD3 cells A. We thu4ZI2) structure with that of the Arl3ΔN∙RP2 complex  although we cannot exclude that the C terminus of BARTL1 might play a role in this interaction. Addition of Arl3 to full-length Cy5-BARTL1 showed no signal change, and therefore could not be used to test for triple complex formation (data not shown). Although BARTL1 does not influence either the intrinsic or the RP2-stimulated GTP hydrolysis of Arl3  and EasyXtal PEG II Suite  from Qiagen. Crystals appeared after 1–3 days and were flash-frozen after 3 days from a 96-well screen in cryosolution containing the same constituents as the crystallizing condition supplemented with 20% glycerol. Crystals from the CORE II Suite were of space group P212121 and crystals from the PEG II Suite were of space group P21 (133 sequence generated by the PHYRE threader based on BART (3DOE) was used in molecular replacement to solve the BARTL1133 structure in the complex. The structure was refined using REFMAC5  and P21 to 2.0 Å . Structures were deposited in the RCSB PDB databank with entry codes PDB: 4ZI2 and 4ZI3, respectively. For data and refinement statistics, see Native full-length Arl3 was purified and exchanged as previously described to be completely loaded with GppNHp . Arl3∙Gproup P21 . Data weroup P21 . Moleculroup P21 . A model REFMAC5  to the f133 was investigated by analytical size-exclusion chromatography using a Superdex200 10/300 column . 0.5 mg of Arl3 protein was incubated with a 10-fold molar excess of GDP or GppNHp for 2 hr at room temperature. The mix was supplemented with 0.5 mg of full-length or truncated BARTL1 or BARTL1133, applied to the size-exclusion chromatography column, and eluted with one column volume of buffer M. The elution profile was recorded and eluted fractions analyzed by SDS-PAGE.Complex formation of Arl3 or Arl3ΔN with BARTL1 or BARTL1133 was shot together with a 50-fold excess of unlabeled BARTL1133. The dissociation of the complex was followed by monitoring the polarization signal at excitation and emission wavelengths of FITC at 490 and 520 nm, respectively. Single exponential functions were fitted to the data using Grafit5 (Erithacus Software) to obtain the koff values.A preformed complex of 2 μM Arl3∙GppNHp with 1 μM FITC-BARTL1WT, Arl3L4D, Arl3F51A, and Arl2WT were loaded with mant-GDP or mant-GppNHp  overnight at 12°C by incubation with a 1.5-fold molar excess of nucleotide, and purified the following day on a Desalting Column in buffer M  to obtain the dissociation constant, KD.Arl3buffer M . Nucleot2 in DMEM/F12, HEPES (Life Technologies) complemented with 10% fetal bovine serum (FBS), and 1% L-glutamine. Stable cell lines were generated as previously described  were cultured at 37°C and 5% COescribed . In shorARL3 or mouse CCDC104 and a negative control siRNA, using Lipofectamine 2000 following the manufacturer’s recommendations. FlexiTube siRNA oligos SI00214963 directed against ARL3, FlexiTube siRNA oligos SI00848855 directed against CCDC104, and negative control siRNA (scrambled) oligo 1027310 were used (Qiagen). 48 hr after transfection of siRNAs against ARL3, cells were serum-starved for 24 hr or, 24 hr after transfection of siRNAs against CCDC104 and direct serum starvation, cells were treated for immunofluorescence microscopy as described below. Images were collected using identical settings for each sample.Stable IMCD3 Flp-In cell lines expressing Arl3 or CCDC104/BARTL1 were plated on poly-L-lysine-coated coverslips. After 24 hr, cells were transfected with 100 nM siRNAs directed against mouse IMCD3 stables expressing GFP fusion proteins were plated on poly-L-lysine-coated coverslips and cilia induced by 48 hr of serum starvation. Cells were washed in PBS and fixed with 4% formaldehyde for 20 min (AcTub) or 2% formaldehyde and 50% ice-cold methanol for 15 min at 4°C (γ-Tub). Cells were permeabilized with 0.3% Triton X-100 in cytoskeletal buffer for 10 min. Cells were rinsed in 0.1% Tween 20 in PBS and blocked in 10% FBS in PBS for 30 min. For immunostaining of primary cilia, mouse 611B1 anti-acetylated α-tubulin antibody (1:5000; Sigma-Aldrich) or anti-Arl13B antibody ; and for basal body staining anti-γ-tubulin antibody  and Arl3 staining anti-Arl3 antibody  in 10% FBS in PBS were incubated overnight at 4°C. Alexa Fluor 647 or 405 anti-mouse or Alexa Fluor 647 anti-rabbit antibody (1:800; Life Technologies) was added for 45 min at room temperature. Coverslips were rinsed three times in 0.1% Tween 20 in PBS and once in PBS. Nuclei were stained with DAPI (Serva) diluted 1:10,000 in PBS for 1 min. Coverslips were fixed on glass slides with Mowiol (Merck). Images were taken using an Olympus IX81 microscope with a CCD camera and a 60× NA 1.35 objective. In all cases at least three independent staining experiments were carried out, and 100 cells were used for analysis.Protein preparation, biochemical/biophysical measurements, generation of stable cell lines, crystallization, X-ray data analysis, and manuscript preparation were carried out by M.L. C.K. cloned various constructs and helped in purifying proteins. S.K. carried out the generation of stable cell lines, knockdown experiments, immunostaining, and analysis by fluorescence microscopy. S.M.L. examined triple complex formation by polarization experiments. TAP experiments were carried out with the help of S.E.C.v.B. TAP eluates were analyzed using mass spectrometry by N.H. and K.B., and data were processed by J.v.R. under supervision of M.U. and R.R., respectively. Project design, supervision, and manuscript preparation were supervised by A.W.