03; KS test) (Figure 2F, G). A summary of these results is shown in Table 1. Figure 2 Effect of silencing several An. gambiae (G3) genes on parasite P. falciparum infection. Effect of silencing arginine kinase (ArgK) (Panel A), heat shock cognate 3 (Hsc-3) (Panel B), solute transporter (Sol. Trsp.) (Panel C), glutathione-S-transferase theta-1 (GSTT1) (Panel D), oxidation resistance gene 1 (OXR1) (Panel E) tetraspanin (Tetrasp.) (Panel F), and glutathione-S-transferase theta-2 (GSTT2) (Panel G) on P. falciparum infection. The number of P. falciparum oocysts

present was determined by directly counting mercurochrome-stained parasites 7–8 days post infection. The dots represent the number Apoptosis inhibitor of parasites present on individual midguts, and the median number of oocysts is indicated by the horizontal line. Distributions are compared using the Kolmogorov-Smirnov test; n = number of mosquitoes; P values lower than 0.05 are considered to be significantly different. Silencing ArgK, Sol. Trsp., and tetraspanin genes has a similar effect on P. berghei and P. falciparum infection. ArgK is a key enzyme in cellular energy homeostasis in arthropods, with a function similar GSK2126458 cell line to that of creatine kinase in mammals. This enzyme catalyzes the synthesis of phosphoarginine, which serves as an energy

reserve. The high-energy phosphate in phosphoarginine can be transferred to ADP to renew ATP during periods of high energy demand [13]. Apparently, silencing this enzyme results in a physiologic state in the mosquito that does not foster the development of either P. berghei or P. falciparum. Silencing of the solute transporter has no effect, while knockdown of tetraspanin enhances infection with both parasites. Tetraspanins Rapamycin mw are proteins with four transmembrane (TM) domains that are associated extensively with one another and with other membrane proteins to form specific microdomains distinct from lipid rafts. They are expressed on the surface of numerous cell types and are involved in diverse processes from cell adhesion to signal transduction and some of them inhibit the function of other members of the same family of proteins

[14]. CD81 is a tetraspanin that has been shown to be required for hepatocyte invasion by P. falciparum and P. yoelii sporozoites [15]. Silencing of the An. gambiae tetraspanin gene may enhance parasite invasion and/or prevent the activation of an immune cascade that limits infection with P. berghei and P. falciparum. OXR1, GSTT1, GSTT2 and Hsc-3 silencing has a different effect on P. berghei and P. falciparum infection. In yeast and mammals, OXR1 is induced by heat and oxidative stress and prevents oxidative damage by an unknown mechanism [16]. In An. gambiae, OXR1 silencing decreases resistance to oxidative challenge and prevents the induction of genes involved in ROS detoxification, such as catalase, following a blood meal (G. Jaramillo-Gutierrez and C. Barillas-Mury, unpublished). We have previously shown that higher ROS levels in An.

The samples were homogenized and sub-samples were diluted in phosphate buffered saline for plating on selective media (MacConkey agar)

supplemented with 100 μg ml-1 streptomycin sulfate. The lower limit of detection in fecal plate counts was 102 CFU (g feces)-1 for 100 μl of the diluted solution per plate. The remaining samples were stored at -80°C. Colony forming units (CFUs) were monitored per gram feces. Phenotypic determination Crude colicin lysates were prepared according to the procedure of Suit et al [42] and stored at 4°C see more until use. Twenty colonies of streptomycin-resistant E. coli from fecal pellets obtained from each mouse at four-week intervals were assayed for the production of growth inhibition zones on plates pre-inoculated with a sensitive lawn (E. coli strain BZB1011). Confirmation of the identity of the colicin produced was provided

by a strain’s ability to grow in the presence of its own colicins (100 μl of crude colicin lysate spread onto LB plates), due to the immunity protein it produces. The zones of inhibition of each strain were documented using an imaging and documentation system (Bio-Rad, Hercules, CA). Statistical analysis Each cage was treated as an independent sample and an average of the two co-caged mice was determined. The average number of CFUs per cage was compared at two times, 0 and 112 days, using a CH5424802 nmr one-way ANOVA. In addition, for each of these times we employed two orthogonal contrasts to test for differences in CFUs among groups of strains that were chosen a priori. One contrast served to compare the average number of CFUs of the colicin-free strain with that of the other (colicinogenic) strains. The second served to compare the average

number of CFUs of the colicinogenic strains. A repeated-measure ANOVA was conducted to test for differences in the persistence of the various strains over time treating strain as a between-subject factor and time as a within-subject factor. The effects of strain type and time (i.e. beginning vs. end of the experiment) on strain doubling time were tested with a two-way ANOVA with both strain and time treated as fixed factors. All statistical analyses were done with the STATISTICA 2007 (StatSoft, Tulsa, OK). Acknowledgements This work was supported by National Institutes of Health grants R01GM068657-01A2 and R01A1064588-01A2 PtdIns(3,4)P2 to M.A. Riley. References 1. Gorbach S, Bartlett JG, Blacklow NR: Infectious Diseases. Philadelphia: Lippincott, Williams, and Wilkins 2003. 2. Guarner F: Enteric flora in health and disease. Digestion 2006,73(Suppl 1):5–12.PubMedCrossRef 3. Altenhoefer A, Oswald S, Sonnenborn U, Enders C, Schulze J, Hacker J, Oelschlaeger TA: The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol Med Microbiol 2004, 40:223–229.PubMedCrossRef 4.

No dominant changes were observed in the optical transmittance spectra after doping, except for the appearance of a slight adsorption around 500 nm by TCNQ molecules [27]. The sheet resistance, R s , as a function of Dasatinib cell line transmittance at 550 nm

is summarized in Figure 7. Due to carrier doping via the CT interaction from TCNQ, the sheet resistance of the RGO + TCNQ complex films drastically decreased by two orders of magnitude without significant degradation of the optical transparency as a result of increasing the sheet carrier density from 1.02 × 1010 cm-2 to 1.17 × 1012 cm-2 estimated from Hall measurement. Doping stability with time evolution at room temperature under ambient atmosphere was monitored. R s increased Staurosporine by less than 10% after 1 year, whereas it increased by up to 40 % after 20 days in the case of AuCl3 which showed one of the highest doping effect [19]. Thermal stability of our doped films was examined by stepwise annealing from 100°C to 250°C under vacuum. The doping effect was preserved after annealing even at 250°C without any remarkable

degradation. This result indicates higher thermal stability than F4-TCNQ [34]. Those stabilities are quite critical issue of doping technique in any application fields. Finally, our chemical doping method was tried by dipping chemical vapor deposition (CVD) graphene purchased from Graphene Platform, Inc. (Houston, TX, USA) in radicalized TCNQ in order to show that our method can be adapted also for CVD graphene. The sheet resistance of the

doped CVD graphene decreased to 400 Ω from 1.2 kΩ at 97% of optical transparency. Our doping method exhibits the compatibility with the CVD graphene-based transparent conductive films. Figure 7 Sheet resistance of different films as a function of optical transmittance at 550 nm. Pristine RGO films (black squares), doped RGO films by surface adsorption (blue triangles), and RGO + TCNQ complex films (red circles). The sheet resistance of the RGO + TCNQ complex films decreased drastically by two orders of magnitude, acetylcholine without degradation of optical transparency, which was a more drastic change than the case of doping by surface adsorption. Conclusions We developed a novel method for the carrier doping of graphene using radical-assisted conjugated organic molecules in the liquid phase. The absorbance data and the Raman spectra results indicated strong charge transfer interactions between RGO and TCNQ. The high doping efficiency of our method was demonstrated as an improvement in sheet resistance by two orders of magnitude, without degradation of the optical transparency. First-principles calculation predicted the model of our doping mechanism and the origin of high doping efficiency. Furthermore, the doping effect was quite chemically stable.

Mol Microbiol 2000,35(4):728–742.PubMedCrossRef 27. Baumler AJ, Tsolis RM, Bowe FA, Kusters JG, Hoffmann S, Heffron F: The pef fimbrial operon of Salmonella typhimurium mediates adhesion to murine small intestine GDC-0449 order and is necessary for fluid accumulation in the infant mouse. Infect Immun 1996,64(1):61–68.PubMed 28. Baumler AJ, Gilde AJ, Tsolis RM, van der Velden AW, Ahmer BM, Heffron F: Contribution of horizontal gene transfer and deletion events to development of distinctive patterns of fimbrial operons during evolution of Salmonella serotypes. J Bacteriol 1997,179(2):317–322.PubMed 29.

Chu C, Chiu CH: Evolution of the virulence plasmids of non-typhoid Salmonella and its association with antimicrobial resistance. Microbes Infect 2006,8(7):1931–1936.PubMedCrossRef 30. Rotger R, Casadesus J: The virulence plasmids of Salmonella . Int Microbiol 1999,2(3):177–184.PubMed 31. Simms AN, Mobley HL: PapX, a P fimbrial operon-encoded inhibitor of buy INK 128 motility in uropathogenic Escherichia coli . Infect Immun 2008,76(11):4833–4841.PubMedCrossRef 32. Li X, Rasko DA, Lockatell CV, Johnson DE, Mobley HL: Repression of bacterial motility by a novel fimbrial gene product. EMBO J 2001,20(17):4854–4862.PubMedCrossRef 33. Clegg S, Hughes KT: FimZ is a molecular link between sticking and swimming in Salmonella enterica serovar Typhimurium.

J Bacteriol 2002,184(4):1209–1213.PubMedCrossRef 34. Tomoyasu T, Takaya A, Isogai E, Yamamoto T: Turnover however of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease. Mol Microbiol 2003,48(2):443–452.PubMedCrossRef 35. Tomljenovic-Berube AM, Mulder DT, Whiteside MD, Brinkman FS, Coombes BK: Identification of the regulatory logic controlling Salmonella pathoadaptation by the SsrA-SsrB two-component system. PLoS Genet 2010,6(3):e1000875.PubMedCrossRef 36. Muller CM, Schneider G, Dobrindt U, Emody L, Hacker J, Uhlin BE: Differential effects and interactions of endogenous

and horizontally acquired H-NS-like proteins in pathogenic Escherichia coli . Mol Microbiol 2010,75(2):280–293.PubMedCrossRef 37. Deighan P, Beloin C, Dorman CJ: Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T. Mol Microbiol 2003,48(5):1401–1416.PubMedCrossRef 38. Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000,97(12):6640–6645.PubMedCrossRef 39. Cummings LA, Wilkerson WD, Bergsbaken T, Cookson BT: In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Mol Microbiol 2006,61(3):795–809.PubMedCrossRef Authors’ contributions LEW, AB and BKC conceived and designed experiments and analyzed data; LEW, AB and BKC performed experiments; LEW and BKC wrote the paper.

Rabbit polyclonal antibodies against lamin A/C as well as mouse monoclonal anti-galectin-3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-villin antibodies were kindly provided by Dr. Sylvie Robine (Curie Institute, Paris). Mouse anti α- tubulin antibodies and rabbit anti-β-catenin antibodies were purchased from Sigma (Munich, Germany). Alexa488 and Alexa546 secondary antibodies were purchased from Invitrogen (Carlsbad, CA). Hoechst 33342 from Fluka (Ronkonkoma, NY)

was used for nuclei staining. 2.2 Kidney sample preparation, cell culture and Western blotting Renal cancer samples, intermediate tissue sample and normal tissue samples of the same Selleckchem Palbociclib kidney were obtained from nephrectomy surgeries. The intersection zone between tumor and normal tissue was defined as intermediate tissue. The study was positively evaluated by the local ethic commission. The patients gave a written informed consent for this study and were not followed clinically. After nephrectomy the specimens were stored in ice-cold PBS

containing a protease Erlotinib purchase inhibitor cocktail and samples were immediately processed for Western blotting, immunohistochemistry or nuclear matrix isolation. Epithelial kidney cells (RC-124) and cells of clear cell renal cell carcinoma (RCC-FG1) (Cell Lines Service, Germany) were cultivated in McCoy’s 5a medium/10% FCS (PAA, Pasching, Austria). Western blot analysis was performed essentially as described before [13]. Protein concentrations were Farnesyltransferase established by Bradford protein assay (BioRad DC Protein Assay, Munich, Germany). Equal amounts of 60 μg/slot were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% skimmed milk powder in PBS. Following immunostaining, bands were detected and quantified using Gel-Pro Software (Kapelan Bio-Imaging, Leipzig, Germany) and normalized to the sum or to tubulin quantities of the same sample. 2.3 Histochemistry and immunohistochemistry Kidney samples from normal, intermediate and tumor tissue were cut into sections of 5 mm and fixed with either formalin (3.7%) or Carnoy (60% Ethanol, 30% chloroform, 10% acetic

acid) overnight and processed as previously described [13]. Images of the samples were captured using a confocal microscope TCS SP2 AOBS (Leica, Wetzlar, Germany). Image stacks were deconvoluted and 3D reconstructed by using the Volocity software package (Improvision, Coventry, UK). 2.4 Nuclear matrix isolation Immediately following nephrectomy, nuclear matrix of homogenized tissues was isolated essentially according to [14]. All procedures were performed on ice and all buffers were cooled to 4°C. Normal and tumor tissue samples from human kidney were Dounce homogenized in 2 ml of buffer A (0.25 M sucrose, 20 mM Tris-HCl, 3 mM MgCl2, pH 7.85 supplemented with a protease inhibitor cocktail) followed by centrifugation at 1000 × g for 10 min at 4°C.

There is an urgent need for clinicians to be able to examine a set of biomarkers such as eIF4E and downstream effector molecules in order to set a current standard for prognosis. Acknowledgements The authors gratefully PF-6463922 in vivo acknowledge the help of Ms. Wanda Green and Dr. Jill Williams in the preparation of the TMAs. The authors also thank the other members of the Breast Cancer Focus Group for helpful discussions on the preparation of this manuscript: Dr. Fleurette Abreo,

Dr. Jun Chung, Dr. Shile Huang, Dr. Kevin Pruitt, Dr. Robert Rhoads, Dr. Amanda Sun, Dr. Songlin Zhang, and Dr. Qian-Jin Zhang. This research was supported by funding from the Feist-Weiller Cancer Center, Shreveport and the Louisiana Gene Therapy Research Consortium. References 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ: Cancer statistics, 2007. CA Cancer J Clin 2007, 57: 43–66.CrossRefPubMed 2. De Benedetti A, Harris AL: eIF4E expression in tumors: its possible role in progression of malignancies. Int J Biochem Cell Biol 1999, 31: 59–72.CrossRefPubMed 3. Dillon RL, White DE, Muller WJ: The phosphatidyl inositol

3-kinase signaling network: implications for human breast cancer. Oncogene 2007, 26: 1338–1345.CrossRefPubMed 4. Santen MAPK Inhibitor Library clinical trial RJ, Song RX, McPherson R, Kumar R, Adam L, Jeng MH, Yue W: The role of mitogen-activated protein (MAP) kinase in breast cancer. J Steroid Biochem Mol Biol 2002, 80: 239–256.CrossRefPubMed 5. Wu JT, Kral JG: The NF-kappaB/IkappaB signaling system: a molecular target in breast cancer therapy. J Surg

Res 2005, 123: 158–169.CrossRefPubMed 6. Sonenberg N: Regulation of translation and cell growth by eIF-4E. Biochimie 1994, 76: 839–846.CrossRefPubMed 7. Richter JD, Sonenberg Methamphetamine N: Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 2005, 433: 477–480.CrossRefPubMed 8. Shantz LM, Pegg AE: Overproduction of ornithine decarboxylase caused by relief of translational repression is associated with neoplastic transformation. Cancer Res 1994, 54: 2313–2316.PubMed 9. Kevil CG, De Benedetti A, Payne DK, Coe LL, Laroux FS, Alexander JS: Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: implications for tumor angiogenesis. Int J Cancer 1996, 65: 785–790.CrossRefPubMed 10. Zimmer SG, DeBenedetti A, Graff JR: Translational control of malignancy: the mRNA cap-binding protein, eIF-4E, as a central regulator of tumor formation, growth, invasion and metastasis. Anticancer Res 2000, 20: 1343–1351.PubMed 11. Rosenwald IB, Lazaris-Karatzas A, Sonenberg N, Schmidt EV: Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E. Mol Cell Biol 1993, 13: 7358–7363.PubMed 12.

The dockings were performed in a 64 bit PC. The receptor design was made by using SWISS-MODEL, a fully automated protein structure homology-modeling server. In this tool, energy minimization and simulated annealing are done with the GROMOS96 forcefield [13]. The 2D structures of the ligands were drawn, optimized with full hydrogen LEE011 bonds and saved as .sk2 format using ChemSketch tool from Advanced Chemistry Development, Inc. (ACD/ChemSketch, [14]) and the 3D structures were obtained using PRODRG server [15]. Receptors The wild type receptor was derived from

the crystal structure of deazaflavin dependent nitroreductase (3R5W) [16]. The mutant receptor was designed by introducing A76E mutation [9], in the sequence of Ddn and modeling it using SWISS MODEL without the presence

of its cofactor F420. Ligands The ligands were derived from the structure of PA-824 by removing the trifluoromethyl group (CF3) and replacing it with key anti-M. tuberculosis drugs such as isoniazid, moxifloxacin, gatifloxacin etc., through ester linkages. The removal of trifluoromethyl group was done on the basis to reduce the toxicity [17]. The designed combinatorial ligands are listed in Table 2. Table 2 Docking values of PA-824 and its novel analogues with the wild and mutant Ddn receptor S.no. Drug Docking score with wild type receptor (kcal/mol) Docking score with A76E mutant receptor (kcal/mol) Structure of the analogues 1 PA-824 −6.9 https://www.selleckchem.com/products/pifithrin-alpha.html −6.7 2 Ligand 1 (glucose) −7.6 −7.0 3 Ligand 2 (Nitroglucose) −7.6 −7.2 4 Ligand 3 (Hydroxyl modification) −6.3 −6.3 5 Ligand 4 (Biotin) −7.1 −6.7 6 Ligand 5 (Cholestryl ester) −8.3 −6.9 7 Ligand 6 (gati) −8.4 −8.1 8 Ligand 7 (isoniazid) −7.8 −7.5 9 Ligand 8 (Moxi) −7.7 −8.5 10 Ligand 9

(PZA) −7.2 −7.2 11 Ligand 10 (Trehalose) −8.0 −7.7 Analysis of binding The binding sites for the docking were designed such that the entire receptor molecule was included within the selection grid. The highest Masitinib (AB1010) binding energy values corresponding to the RMSD value of zero were considered as the binding affinity value of the ligands for each docking. The Hydrogen bond interactions were obtained using Molegro molecular viewer (molegro.com) [18]. Results Bactericidal activity The results of the bactericidal activity of different drugs from the two sets of experiments are given in Figure 1. PA-824 at lower concentration of 3 μg/ml (P1) had less activity on all the days resulting with a log of 4.9 CFU/ml on the 21st day. Rifampicin (1 μg/ml) showed slightly increased activity than PA-824 at a lower concentration of 3 μg/ml, with a reduction in the count of 1.42 log cfu/ml on the 7th day, whereas for PA-824 at a concentration of 12.5 μg/ml (P2), showed a decrease in the count to log of 2.49 CFU/ml on the same day. A small reduction in RIF activity was seen on the 7th day, and on 14th day reduction of 2.

So, improvement of existing methods or development of new methods is needed for the analysis of gene expression microarray data. Many gene expression signatures have been identified in recent years for accurate classification of tumor

subtypes [16–19]. It has been indicated that rational use of the available bioinformation can not only effectively remove or suppress noise in gene chips, but also avoid one-sided results of separate experiment. However, a relatively few attempts have been aware of the importance of prior information in cancer classification [20–22]. Lung cancer is one of the leading causes of cancer death worldwide [23–26], can be classified broadly into small cell lung buy 5-Fluoracil cancer (SCLC) and non-small cell lung cancer (NSCLC), and adenocarcinoma

is the most common form of lung cancer. Because in China the cigarette smoking rate continues to be at a high level [27], a peak in lung cancer incidence is still expected [28]. Therefore, only lung cancer gene expression microarray dataset was selected in the present study. In summary, together with the application of support vector machine as the discriminant approach and PAM as the feature gene selection method, we selleck propose one method that incorporates prior knowledge into cancer classification based on gene expression data. Our goal is to improve classification accuracy

based on the publicly available lung cancer microarray dataset [29]. Methods Microarray dataset In the present study, we analyzed Ribonucleotide reductase the well-known and publicly available microarray dataset, malignant pleural mesothelioma and lung adenocarcinoma gene expression database http://​www.​chestsurg.​org/​publications/​2002-microarray.​aspx[29]. This Affymetrix Human GeneAtlas U95Av2 microarray dataset contains 12 533 genes’ expression profiles of 31 malignant pleural mesothelioma (MPM) and 150 lung adenocarcinomas (ADCA, published in a previous study [30]), aims to test expression ratio-based analysis to differentiating between MPM and lung cancer. In this dataset, a training set consisted of 16 ADCA and 16 MPM samples. Microarray data preprocessing The absolute values of the raw data were used, then they were normalized by natural logarithm transformation. This preprocessing procedure was performed by using R statistical software version 2.80 (R foundation for Statistical Computer, Vienna, Austria). Gene selection via PAM Prediction analysis for microarrays (PAM, also known as Nearest Shrunken Centroids) is a clustering technique used for classification, it uses gene expression data to calculate the shrunken centroid for each class and then predicts which class an unknown sample would fall into based on the nearest shrunken centroid.

After 1 h of incubation at 37 °C in the dark, the reaction mixtures were mixed with 4 mL of loading buffer (bromophenol blue in 30 % glycerol) and selleck loaded on 1 % agarose gels containing ethidium bromide (Sigma-Aldrich), in TBE buffer (90 mM Tris–borate, pH 8.0; 20 mM EDTA). Gel electrophoresis was done at a constant voltage of 4 V/cm for 60 min. As a control for double-strand breaks, reference plasmid samples were linearized with EcoRI endonuclease. The gels were photographed and processed with a Digital Imaging System (Syngen Biotech, Wroclaw, Poland). Reactive oxygen

species (ROS) generation measurements The ROS generation measurements were carried out with NDMA (N,N-dimethyl-4-nitrosoaniline) and NBT (nitrotetrazolium blue chloride), a scavenger molecules commonly used in studies of hydroxyl radicals and superoxide anion generation, respectively. The experiments were followed at 25 °C on a Cary 60 spectrophotometer. PF-02341066 concentration The solutions of NDMA and NBT at final concentrations 20 μM were added to the samples containing 50 μM Cu(II), MTX and Cu(II)–MTX,

in the presence of 50 μM H2O2, at pH 7.4 (0.2 M phosphate buffer). The generation of singlet oxygen was tested by gel electrophoresis in conditions described above (“DNA strand break analysis” section) with an extra addition of NaN3 (singlet oxygen scavenger (Franco et al., 2007)) at final concentration 40 mM. Cytotoxic assay Cell lines and culture conditions CT26 cell line (mouse colon carcinoma, morphology: fibroblast, ATCC: CRL–2638) and A549 cell line (human lung adenocarcinoma, morphology: epithelial, ATCC: CCL–185) were obtained from professor Luis G. Arnaut group (Chemistry Department, University of Coimbra, Portugal). Cells were cultured in flasks in Dulbecco’s Modified Eagle Medium (DMEM) without phenol red, with 10 % fetal bovine serum (FBS) and with 1 % streptomycin/penicillin at 37 °C and 5 % CO2 in a humidified atmosphere. Cells were passaged at preconfluent densities, using a solution containing 0.05 % trypsin and 0.5 mM EDTA. All the cell culture

fluids were purchased from IMMUNIQ (Poland). Cytotoxicity study The cytotoxic activity in vitro was evaluated by the MTT assay. The assay was carried out according to the well-known protocol (Slater et al., 1963). For the screening experiments, exponentially oxyclozanide growing cells were harvested and plated in 96–well plates at a concentration of 1 × 104 cells/well. After 24 h of incubation at 37 °C under humidified 5 % CO2 allowing cell attachment, the cells in the wells were treated with tested compounds at various concentrations in the range from 1 to 100 μM. The compounds were predissolved in phosphate buffer (pH 7.4) and diluted in the respective medium with 1 % FBS. Two different protocols of cytotoxicity evaluation were performed. In the first approach cells were treated with 200 μL of tested samples: CuCl2, MTX, Cu(II)–MTX, and cisplatin for 4 h at 37 °C under conditions of 5 % CO2.

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