Ralstonia accounted for a Epoxomicin cost significantly greater proportion of the bacterial community in the surface sterilized samples, implying that it was largely endophytic, and was also a significantly lower proportion of the community in the samples from organically grown varieties. Acinetobacter accounted for a significantly lower proportion of the community in surface sterilized MK-2206 purchase samples, suggesting that it was primarily associated with the leaf surface. Table 2 Dominant members of bacterial communities associated with leafy salad vegetables as determined

from pyrosequencing Genus (or higher) Baby spinach Romaine lettuce Red leaf lettuce Iceberg lettuce Green leaf lettuce C Cs O Os C Cs O Os C Cs O Os C Cs O Os C Cs O Os Pseudomonas 93.8 70.6 40.5 20.7 23.9 67.0 67.2 36.1 76.3 18.9 54.7 27.4 11.1 7.1 2.5 59.9 28.7 33.2 5.1 15.0 Ralstonia *(S, O) – - – - – - – - 11.8 76.5 1.6 Pritelivir concentration 38.7 14.7 82.7 0.7 20.4 60.7 60.3 – 53.4 Flavobacterium 1.5 8.9 38.9 72.1 1.1 0.5 – 0.3 0.2 0.1 18.5 7.3 3.6 0.3 – 9.4 0.3 0.1 2.0 0.5 Stenotrophomonas – 2.3 0.1 2.8 20.2 20.0 30.8 62.2 – 0.1 – 0.2 1.9 0.5 1.0 1.3 0.5 1.1 – 0.3 Serratia 1.2 0.2 – 0.1 – - – - – - 0.1 1.3 5.1 3.7 – 0.7 0.3 – 66.0 18.6 Erwinia 1.9 10.5 – 0.1 0.2 – 0.1 – 0.1 – - – 1.3 0.2 58.6 0.8 0.3 – 0.4 0.1 Xanthomonas – - – - 47.4 – 0.1 – - – - – 51.4 0.5 – - – - – - Pantoea 0.1 1.4 0.1 0.1 1.0 3.0 – 0.1 0.1 0.1 – 0.1 1.1 0.1 17.6 1.1 1.1 0.6 0.1 0.3 Providencia – - – - – - – - – - – 0.1 0.8 0.5 – - – - 13.9 3.9 Enterobacteriaceae unk.. 0.8 0.9 1.0 0.2 2.1 0.5 0.7 0.4 0.3 0.1 1.3 0.4 2.1 0.3 0.5 0.6 0.6 0.2 0.8 1.8 Janthinobacterium 0.2 2.9 1.2 0.2 0.4 – - – 0.1 – 7.6 Rebamipide 4.1 0.3 0.2 – 0.3 0.3 0.1 0.8 0.5 Shewanella – - 13.1 0.4 – - 0.1 – - – - – - – - – - – - – Enterobacter 0.1 0.2

– 0.3 – 0.4 – - 0.5 0.3 – 0.5 1.4 0.6 2.4 – - – 2.6 1.3 Enhydrobacter – - – - 0.1 – - – 2.3 – 3.4 3.5 0.1 – - – - – 0.3 0.3 Leeia – - – - – - – - 1.2 1.0 – 1.5 0.1 0.5 – 1.3 1.3 0.9 – 0.8 Morganella – - – - – - – - – - – 8.5 – - – - – - – - Massilia *(S) – - 0.1 0.1 0.2 – - – 1.3 – 1.7 1.3 0.4 0.1 – 0.2 0.2 0.1 0.2 0.1 Duganella 0.1 – - – - – - – 0.4 – 3.5 0.9 0.1 – 0.2 0.1 0.1 – - – Acinetobacter *(S) – - 0.8 – 0.2 – - – 0.1 – 0.5 0.1 0.5 – 0.4 0.1 – - 0.6 0.2 Bacillus – - – - – 3.4 – - – 0.2 – - – - – - – - – - Streptococcus – - 0.2 1.5 0.1 0.1 – - – - – - – 0.4 – - 0.1 0.1 – - Staphylococcus – - 0.3 0.4 0.3 0.1 – - – - – - 1.1 – - – 0.5 – - – Chryseobacterium – 0.2 0.9 – - 0.2 – - – - 0.2 – 0.1 – 0.4 – - – 0.

Peak shifts at large T indicate the extent of static disorder, and the decay captures population dynamics.

For example, Jimenez et al. (1997) find more revealed that initial peak shifts for light-harvesting complexes (LH1 and LH2) of purple photosynthetic bacteria, Rhodobacter (Rb.) sphaeroides are large (~25 fs) compared to the peak shifts of typical dyes in polar solvents (10–15 fs), which indicates weak coupling of the pigments in these complexes to the surrounding protein matrix. This relatively weak coupling may be essential to minimize heat dissipation to the surroundings and, therefore, maximize the energy transfer efficiency from LH2 to LH1 to the reaction center. Another 1C3PEPS experiment on the isolated B820 subunit (a subunit of the LH1 complex, so-called because it absorbs near 820 nm) of LH1 in Rhodospirillum rubrum, in comparison with 1C3PEPS on the whole LH1 complex, clearly demonstrated the contribution MK0683 mw of energy transfer to the 1C3PEPS signal decay (Fig. 3) (Yu et al. 1997). The signal from the

LH1 complex showed a rapid decay component in early T corresponding to energy transfer around the ring and resulting in a small peak shift value at long T (circles). Note that (excitation) energy transfer from one (excited) molecule MX69 cell line to another leads to loss of correlation. To the contrary, the energy transfer out of the subunit is blocked in the B820 subunit, which consists only of one α and one β transmembrane polypeptide and two BChla molecules. Therefore, the B820 subunit exhibits a generally large peak shift (squares, Fig. 3). The solid line indicates the simulated 1C3PEPS profile with Decitabine price the same parameters for the LH1 complex but without an energy transfer factor.

The experiments also demonstrate that the photon echo peak shift is sensitive to energy transfer within the laser pulse window as well as energy transfer out of the detection window because the peak shift measures the rephasing capability. Moreover, unlike conventional transient absorption or time-resolved fluorescence studies, it is insensitive to reverse energy transfer between transitions of similar energies. These features are useful in studying the diagonal elements of a Hamiltonian of photosynthetic systems in which multiple replicas of pigments are common. In this sense, the evolution of photon echo peak shift reflects excited state dynamics of a photosynthetic system in detail. Fig. 3 1C3PEPS measurements of LH1 of Rhodobacter (Rb.) sphaeroides (circles) and the B820 subunit from LH1 of Rhodospirillum (Rs.) rubrum (squares). The solid lines represent two simulations with identical input parameters except that the energy transfer rate is set to zero for the B820 sample (Yu et al. 1997). Figure reprinted by permission from Elsevier (Yu et al.

As shown ACP-196 mw in Figure 1, the adhesion to fibronectin was differentially modulated by the antibiotics. Oxacillin-, moxifloxacin-, clindamycin- and linezolid-treated bacteria displayed increased binding to fibronectin. This effect was observed for all strains tested except fnbA/B-negative DU5883. The increase in amplitude of fibronectin binding was strain-dependent. Oxacillin treatment increased fibronectin binding from 1.8- to 2.7-fold relative to the untreated control; moxifloxacin treatment increased binding from 1.4- to 2.3-fold; clindamycin

treatment increased binding from 1.5- to 1.8-fold; and linezolid treatment increased binding from 1.6- to 2.3-fold, depending on the tested strain. By contrast, fibronectin binding was significantly reduced after rifampicin treatment. The decrease was strain-dependent and ranged from 1.5- to 3.5-fold compared to the untreated control. Vancomycin and gentamicin had no effect on bacterial adhesion to fibronectin-coated plates (data not shown). 4SC-202 mw Antibiotics-induced reduction in bacterial density had no significant confounding effect on fibronectin binding in our model, as demonstrated by the absence of correlation between n-fold changes in bacterial density and fibronectin binding in antibiotics-treated NVP-LDE225 datasheet strain 8325-4 (Additional File 1). The DU5883 strain, defective for fnbA and fnbB genes [9], did not adhere to fibronectin-coated

plates in any condition (with or without antibiotics). Clindamycin could not be tested with the DU5883 strain as it harbours the ermB gene and therefore is resistant to clindamycin (Table 3). Figure 1 Effect of antibiotics on the adhesion to human fibronectin. Exponential growth

Acyl CoA dehydrogenase cultures of S. aureus laboratory strains 8325-4 and DU5883 and clinical isolates ST2008 1028, ST2008 0563, HT2000 0594 and HT2001 0390 were treated or not treated with 1/2 of the MIC of antibiotics (oxacillin, moxifloxacin, clindamycin, linezolid or rifampicin) and assayed for adhesion to fibronectin-coated microplates, as described in Methods section. The results are OD570 nm values reflecting bacterial adhesion to fibronectin. The values were obtained from 3 different wells previously incubated with the same bacterial suspension, and adhesion is expressed as the mean ± standard deviation (dark bars for untreated cultures and white bars for antibiotic treated cultures; results from three different experiments). Asterisk = significantly different from the control (corresponding isolate grown without antibiotic), with a P value of 0.05 by one-way analysis of variance followed by a posteriori Dunnett’s test. Effect of antibiotics on fnbA and fnbB mRNA levels We explored the effect of antibiotics on mRNA expression levels of the fnbA and fnbB genes which encode FnBPA/B. The fnbA and fnbB mRNA levels in exponential phase cultures of S.

(Meanwhile, one night during the winter, 1 week after Loeb had arrived for a vacation in Bermuda, Jacques Loeb died at the Biological Station in the room that was just above Blinks’s room.) Blinks had collaborated with Osterhout and Loeb in critical membrane transport work at the Rockefeller Institute and at the Bermuda Biological Station in the 1920s. This work included some of the earliest measurements of ion transport across cell membranes, of membrane conductance and transmembrane electric potential. The work formed the

basis of our understanding of electrical activities in cells and was incorporated into animal research as well as plant physiology (Briggs et al. 1990). Blinks measured the fundamental parameters of the environmental find more variability of algal cells such as pH, various concentrations of the major ionic salts, temperature, IWR1 pressure, and light to elucidate the environmental variables acting on algal cells versus Stattic cell line their electric characteristics (Blinks 1928, 1929, 1933, 1936a, b). He continued working with Osterhout into the early 1930s. At this time, the Great Depression hit the Rockefeller Institute’s funding. For Blinks, a more serious problem was that

Winthrop Osterhout suffered a massive heart attack in the winter of 1931. Blinks had previously been courted by Stanford University for a faculty position and been asked to teach at a summer session at Stanford. Upon Osterhout’s illness, Stanford offered Blinks a position in 1931. Blinks moves to Stanford and begins photosynthesis research Blinks was an associate professor and eventually a full professor at Stanford University’s main campus from 1931 to 1943. During 1943–1964, Blinks served as the Director of Stanford’s Hopkins Marine Station (Pacific Grove). In 1955, he was elected a member of the

National Academy of Sciences, USA. He left Stanford only five times: (1) for a year as Vice President (1954–1955) of Interleukin-3 receptor the National Science Foundation under William McElroy’s presidency; (2) for a sabbatical (1940–1941) ostensibly at the Carnegie Institutes’ Tortugas Marine Laboratories (which was unavailable during World War II, so he stayed in Key West, Florida to study giant marine plant cell membranes); (3) another sabbatical in Stockholm, Sweden at the Nobel Institute; (4) a third sabbatical in 1949 in Cambridge, England on a Guggenheim award; and (5) at age 65, upon retirement from Stanford, Blinks also participated in the building of the Department of Biological Sciences at the University of California, Santa Cruz (1965–1973).

9)], the SabR-His6 proteins were specifically eluted from the resin with 4 ml elution buffer [20 mM Tris base, 500 mM NaCl, 250 mM imidazole, 5 % glycerol (pH 7.9)] and concentrated to about 20 μg μl-1 by ultrafiltration (Millipore membrane, 3 kDa cut-off size) according to the protocol provided by the manufacturer. Protein purity was determined

by Coomassie brilliant blue staining after SDS-PAGE on a 12 % polyacrylamide gel. The purified protein was stored in 5 % glycerol at -70°C. Electrophoretic mobility-shift assays (EMSAs) The EMSAs were performed as described previously [37]. The primers were labeled with T4 DNA polynucleotide kinase and the DNA fragments used for [γ-32P]-labeled probes were amplified by PCR, and then purified by using

PCR purification kit (Qiagen). For EMSAs with SabR-His6, the sanG probes were generated by PCR using primers EG0-F, EG1-F, EG2-F, EG3-F and EG0-R, EG1-R, NU7441 clinical trial EG2-R, EG3-R, which were uniquely labeled at its 5′ end with [γ-32P]-ATP using T4 polynucleotide kinase respectively. The sabR, sanF and sanNO probes were generated by PCR using unlabeled primers ER-F, EF-F, ENO-F and the radiolabeled PF-6463922 molecular weight primers ER-R, EF-R and ENO-R, respectively. During the EMSA, the [γ-32P]-labeled DNA probe (1000 cpm) was incubated individually with varying quantities of SabR-His6 at 25°C for 25 min in a buffer containing 1 μg of poly-(dI-dC) (Sigma), 20 mM Tris-base (pH 7.5), 1 mM DTT, 10 mM MgCl2, SB-3CT 0.5 μg calf BSA μl-1 and 5 % glycerol in a total volume of 20 μl. After incubation, protein-DNA complex and free DNA were separated by electrophoresis on non-denaturing 4.5 % polyacrylamide gels with a running buffer containing 45 mM Tris-HCl (pH 8.0), 45 mM boric acid and 1 mM EDTA at 10 V cm-1 and 4°C. Gels were dried and exposed to Biomax radiographic film (Kodak). As controls, unlabeled probe (25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 175-fold and click here 200-fold specific competitor or 25-fold, 50-fold, 100-fold and 200-fold non-specific competitor) and labeled probe were mixed with SabR-His6 and incubated for 25 min at 25°C. The resulting DNA-protein complexes were then subjected

to electrophoresis and autoradiography as described above. In order to quantify all probes, the probe DNA concentration was detected by ultraviolet spectrophotometer at the wavelength of 260 nm. DNase 1 footprinting To characterize the SabR-binding sites upstream region of sanG, a DNA fragment was amplified by PCR with the labeled primer EG1-F. The footprinting reaction mixture contained 30,000 cpm of [γ-32P]-labeled DNA probe, 6 ng to 0.3 μg of SabR-His6, 2.5 μg of poly-(dI-dC) (Sigma) and 20 mM Tris-base (pH 7.5), 1 mM DTT, 10 mM MgCl2, 0.5 μg calf BSA μl-1 and 5 % (v/v) glycerol in a total volume of 50 μl. After incubation of the mixture at 25°C for 25 min, 5.5 μl RQ1 RNase-free DNase Buffer and 0.1 U DNase 1 were added to the above reaction and the mixture was incubated for 1 min.

Altern Med Rev 2009,14(2):154–60.PubMed AZD6738 304. Maki KC, Reeves MS, Farmer M, Yasunaga K, Matsuo N, Katsuragi Y, Komikado M, Tokimitsu I, Wilder D, Jones F, Blumberg JB, Cartwright Y: Green tea catechin consumption enhances exercise-induced abdominal fat loss in overweight and obese

adults. J Nutr 2009,139(2):264–70.PubMed 305. Fallon E, Zhong L, Furne JK, Levitt M: A mixture of extracts of black and green teas and mulberry leaf did not reduce weight gain in rats fed a high-fat diet. Altern Med Rev 2008,13(1):43–9.PubMed 306. Hsu CH, Tsai TH, Kao YH, Hwang KC, Tseng TY, Chou P: Effect of green tea extract on obese women: a randomized, double-blind, placebo-controlled clinical trial. Clin Nutr 2008,27(3):363–70.PubMedCrossRef 307. MacDonald HB: Conjugated linoleic acid and disease prevention: a review of current knowledge. J Am Coll Nutr 2000,19(2 Suppl):111S-8S.PubMed 308. Park Y, Albright KJ, Storkson JM, Liu W, Cook ME, Pariza MW: Changes in body selleck chemicals composition in mice selleck screening library during feeding and withdrawal of conjugated linoleic acid. Lipids 1999,34(3):243–8.PubMedCrossRef

309. Colakoglu S, Colakoglu M, Taneli F, Cetinoz F, Turkmen M: Cumulative effects of conjugated linoleic acid and exercise on endurance development, body composition, serum leptin and insulin levels. J Sports Med Phys Fitness 2006,46(4):570–7.PubMed 310. Lowery LM, Appicelli PA, PWR L: Conjugated linoleic acid enhances muscle size and strength gains in novice bodybuilders. Med Sci Sports Exerc 1998,30(5):S182. 311. Riserus U, Arner P, Brismar K, Vessby B: Treatment with dietary trans10cis12 conjugated linoleic acid causes isomer-specific insulin resistance in obese men with the metabolic syndrome. Diabetes Care 2002,25(9):1516–21.PubMedCrossRef 312. Riserus U, Basu S, Jovinge Resminostat S, Fredrikson GN, Arnlov J, Vessby B: Supplementation with conjugated linoleic acid causes isomer-dependent oxidative stress and elevated C-reactive protein: a potential link to fatty

acid-induced insulin resistance. Circulation 2002,106(15):1925–9.PubMedCrossRef 313. Riserus U, Berglund L, Vessby B: Conjugated linoleic acid (CLA) reduced abdominal adipose tissue in obese middle-aged men with signs of the metabolic syndrome: a randomised controlled trial. Int J Obes Relat Metab Disord 2001,25(8):1129–35.PubMedCrossRef 314. Thom E, Wadstein J, Gudmundsen O: Conjugated linoleic acid reduces body fat in healthy exercising humans. J Int Med Res 2001,29(5):392–6.PubMed 315. Cornish SM, Candow DG, Jantz NT, Chilibeck PD, Little JP, Forbes S, Abeysekara S, Zello GA: Conjugated linoleic acid combined with creatine monohydrate and whey protein supplementation during strength training. Int J Sport Nutr Exerc Metab 2009,19(1):79–96.PubMed 316. Beuker F, Haak H, Schwietz H, editors: CLA and body styling. Symposium: Vitamine und Zusatzstoffe; Jena (Thhr.) 1999. 317.

7 mm dia. pins that U0126 solubility dmso deliver 0.34 μl each. Before and between applications pins were cleaned by submersion

in 10% bleach and 70% ethanol for 5 s each followed by drying for 10 s with warm sterile air. The plates were incubated at 30°C for 48 h and halos were verified by visual inspection. Growth inhibition click here measurement in liquid culture Yeast strains (OD600 = 0.02) were incubated with appropriate dilutions of each compound in 200 μl cultures in 96-well plates, in addition to DMSO controls. Kinetic growth curves were generated with a TECAN plate reader by reading the OD every 2 h after agitating the plate prior to reading to suspend the yeast. For growth comparisons between different treatments the exponential part of the growth curve was considered and ODs were transformed into log10 values. The least squares method was applied to generate a straight line that best fit the data and line slopes were calculated to compare growth behaviour between different growth conditions. Drug dosage suppressor screen Multicopy pool construction and growth – A S.

cerevisiae random genomic library AZD8931 (gift from Martha Cyert) constructed in a high-copy 2 micron expression vector (YEplac195) with an average insert size of approximately 5 kb was transformed into yeast (BY4743) by a standard lithium acetate method [52] and selected in -URA dropout medium. After 3 days incubation at 30°C, ~106 transformants were pooled into medium containing 7% DMSO, aliquoted, and stored at -80°C. For screens, frozen aliquots were thawed and inoculated directly into 700 μl -URA dropout medium to an OD600 = 0.02. Compound was added and the pool was grown for 5 generations in 48-well microtiter plates (Nunc). Final compound concentrations were as follows: 50

μM for dhMotC, analogue 20 and 27, 6 μM for analogue 21. An inhibitory concentration of at least 50% (IC50) was necessary to provide sufficient selection when screens were performed for 5 generations. Cells were harvested automatically by a Packard Multiprobe II four-probe liquid-handling system (PerkinElmer). Plasmid isolation, insert PCR amplification and microarray hybridization – Plasmids were isolated using the Zymoprep PTK6 II plasmid isolation kit (Zymo Research). The inserts were amplified by PCR with the FailSafe™ PCR System (Epicentre® Biotechnologies) using common M13 primers. PCR cycling conditions were: an initial melting step at 95°C for 2 min followed by 30 cycles at 95°C for 0.5 min, 58°C for 0.5 min and 68°C for 10 min followed by a final extension at 68°C for 15 min. The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and labelled with biotin using the BioPrime labelling kit (Invitrogen). Labelled products were hybridized to Affymetrix TAG4 arrays using the same protocols as described for TAG hybridizations [53]. Multicopy suppression profiling (MSP) analysis – ORF probe intensities were extracted and normalized.

For quantitative analysis,

western blot signals were normalized against total Z-VAD-FMK price proteins detected per lane in the corresponding MemCode stained membrane using the QuantityOne software (not shown). Figure 6 Western blot analysis of cadherin MCC950 protein expression. (A) Percent index variance analysis of the western blot showing cadherin expression: (C1) 2-day old uninfected cultures, (C2) 3-day old uninfected SkMC (control), (I) cultures after 24 h of interaction with T. gondii tachyzoites, and (P) parasites alone (confirming the absence of synthesis of cadherin by T. gondii tachyzoites). Quantitative analysis revealed only 10% reduction in the expression of cadherin between normal cultures, reaching values of more than 50% reduction in T. gondii infected SkMC after 24 h. Results are representative of three independent experiments. Student’s T-test (*) p ≤ 0.05. RT-PCR analysis of M-cadherin

mRNA in SkMC- T. gondii infected cells M-cadherin gene expression in SkMC experimentally infected with T. gondii was analyzed by RT-PCR. M-cadherin mRNA was detected 2 and 3 days after plating and it was up regulated only after the induction of myotube formation, which corresponds to the second day of culture. After 3 h see more of infection with T. gondii M-cadherin mRNA levels were significantly reduced and after 12 h of interaction, no change in M-cadherin mRNA expression profile was observed. However, after 24 h, M-cadherin mRNA expression was down regulated when compared to the corresponding SkMC control from 3 day-old cell cultures aminophylline (Figure 7A-C). Figure 7 Profile of M-cadherin mRNA expression by SkMC experimentally infected with T. gondii. (A) The arbitrary values presented in the graph are

based on the densytometric analysis of the PCR gel image shown in panel B, corresponding to 3, 12 and 24 h of infection. Light bars indicate uninfected control cells and black bars indicate the infected cells. (B) Polyacrylamide, silver stained gels for visualization of the amplified M-cadherin and GAPDH mRNAs (from top to bottom, respectively). Lanes 1, 3 and 5 show the profiles of negative controls and lanes 2, 4 and 6 the profiles of infected cells (3, 12 and 24 h, respectively). NC, negative PCR control. Molecular size markers are indicated to the left. Student’s T-test (*) p ≤ 0.05. Discussion This study analyzes the impact of T. gondii-infection on the myogenesis process. The results obtained showed that: (i) myoblasts are more susceptible to infection than myotubes; (ii) T. gondii-infected myoblasts are unable to fuse with others myoblasts and myotubes and, (iii) M-cadherin expression is down regulated during infection, indicating that T. gondii interferes with myogenesis in SkMC model. We have observed that after 24 h of T. gondii-SkMC interaction, myoblasts are more infected than myotubes.

The MIC was defined as the lowest concentration of metal that allowed no bacterial growth. For each metal and bacterial strain, at least three independent experiments were carried out. β-galactosidase assay Enzyme activities were measured from bacteria grown overnight in LB or in LB supplemented with different metal salts (concentrations are specified in Results). β-galactosidase activity was assayed according to a previously described protocol [68]. Western blotting Cell lysates were prepared from bacteria grown overnight in LB or in LB supplemented with either 0.6 mM ZnSO4

or 0.15 mM FeSO4. Equal amounts of total protein (3 μg) were separated by Tricine-SDS-PA gel electrophoresis, followed by protein transfer to a nitrocellulose membrane. For Western blotting, the membranes were probed with ColR-specific polyclonal antibodies, followed by treatment selleck kinase inhibitor with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G. The blots

were developed using bromochloroindolyl phosphate/nitro blue tetrazolium (BCIP/NBT). Acknowledgments We are grateful to Hedvig Tamman, Andres Ainelo and Hanna Hõrak for critically reading the manuscript. We thank Külliki Holtsmann for assistance in the cloning and Peeter Hõrak and Riho Teras for advice in the statistical analysis. This work was supported by the grant 7829 from the Estonian Science Foundation, by Targeted Financing Project TLOMR0031 and by Institutional Selleckchem Belinostat Research grant IUT20-19.

Electronic supplementary material Additional file 1: Table S1: Bacterial strains and plasmids. (DOCX 56 KB) Additional file 2: Table S2: The oligonucleotides. (DOCX 22 KB) Additional file 3: Table S3: The oligonucleotide pairs used in two sequential PCRs for site-directed mutagenesis of colS. (DOCX 18 KB) References 1. Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM: Metal ions in biological catalysis: from enzyme databases to general principles. J Biol Inorg Chem 2008,13(8):1205–1218.CrossRef 2. Touati D: Iron and oxidative stress in bacteria. Arch Biochem Biophys 2000,373(1):1–6.PubMedCrossRef 3. Imlay JA: Iron-sulphur Ribose-5-phosphate isomerase clusters and the problem with oxygen. Mol Microbiol 2006,59(4):1073–1082.PubMedCrossRef 4. McDevitt CA, Ogunniyi AD, Valkov E, Lawrence MC, Kobe B, McEwan AG, Paton JC: A molecular mechanism for bacterial susceptibility to zinc. PLoS Pathog 2011,7(11):e1002357.PubMedCentralPubMedCrossRef 5. Outten CE, O’Halloran TV: Femtomolar sensitivity of metalloregulatory find more proteins controlling zinc homeostasis. Science 2001,292(5526):2488–2492.PubMedCrossRef 6. Changela A, Chen K, Xue Y, Holschen J, Outten CE, O’Halloran TV, Mondragon A: Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 2003,301(5638):1383–1387.PubMedCrossRef 7. Nies DH: Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 2003,27(2–3):313–339.PubMedCrossRef 8.

600 μl RPMI1640 containing 20% FBS was added to the lower chamber. After the cells were

incubated for 72 h (invasion) or 36 h (migration) at 37°C in a 5% CO2 incubator, the cells on the top surface of the insert were removed by wiping with a cotton swab. The cells that migrated to the bottom surface of the insert were fixed in 100% methanol for 2 min, stained in Giemsa for 2 min, rinsed in PBS and then subjected to microscopic inspection (×200). Values for invasion and migration were obtained by counting five fields per membrane and represented the DAPT clinical trial average of three independent experiments [12]. Statistics analysis The data were presented as means-standard errors (SE) for MDA-MB-231 cells in each group. Statistical analysis was carried out by one-way ANOVA followed by Dunnett t-test or Student t-test (two means comparison). Statistical analysis was given using the related programs in SPSS 12.0. Differences were considered significant when P < 0.05. Results check details JMJD2A siRNA synthesis The sequence of chemically synthesized JMJD2A siRNA was consistent with the requirements, and the purity reached

to 98%. This met the experiment requirements. Observation EX 527 manufacturer of cell transfection results MDA-MB-231 cells transfected with FAM-siRNA were subjected to Fluorescence microscopy at 8 h after transfection. The green fluorescence cells were considered to be transfected successfully. As shown in Figure 1A, cell transfection was successful and HiPerFect Transfection Reagent was effective. The transfection efficiency was about 72.3%. Figure 1 Transfection was successful and levels of JMJD2A mRNA and protein were both down-regulated. A. The green fluorescence cells transfected with FAM-siRNA under fluorescence microscope (Note: ×100). B. Column diagram analysis for mRNA levels of JMJD2A. JMJD2A-specific siRNA resulted in the reduction of JMJD2A mRNA levels in MDA-MB-231 cells. C. Western blot analysis for JMJD2A protein. D. Column diagram analysis for optical density by Western blotting. JMJD2A protein levels were down-regulated

in siRNA group. (*P < 0.05, compared with blank control group and negative control group respectively) Transfection with JMJD2A-specific siRNA down-regulated JMJD2A mRNA levels to silence JMJD2A gene out According to the results of quantitative real-time PCR (Figure 1B), no significant difference (P > 0.05) was detected in the levels of JMJD2A mRNA between blank control group (0.998 ± 0.170) and negative control group (0.997 ± 0.150). The mRNA expression of siRNA group (0.386 ± 0.108) were significantly lower than that in blank control group (P < 0.05) and negative control group (P < 0.05), respectively. These data suggested that JMJD2A mRNA levels in MDA-MB-231 cells decreased significantly after transfection with JMJD2A siRNA. Transfection with JMJD2A-specific siRNA could result in JMJD2A mRNA degradation to silence JMJD2A gene.