Then, absorption of samples was measured at 562 nm in a Perkin Elmer Lambda 25 UV/Vis spectrophotometer and compared to protein standards containing bovine serum albumin in a concentration range of 0-600 μg ml-1. Extraction selleckchem and determination of intracellular trehalose content Trehalose determination was performed basically as described by Blázquez et al. [52] by the following procedure. Cell pellets from 15 ml of early stationary phase cultures in their optimal minimal medium were washed with isotonic carbon-free medium and resuspended in 1 ml of the same medium. Cells were lysed by 30 min incubation at 95°C and, after

centrifugation, trehalose was assayed in a 200 μl total volume reaction containing 100 μl of the supernatant,

90 μl of 25 mM sodium acetate buffer (pH 5.6) and 0.02 U of commercial trehalase (Sigma). For each culture sample, endogenous glucose content was monitored by performing a parallel reaction in which trehalase was substituted by water. After overnight incubation at 37°C, glucose released by trehalose hydrolysis was determined on 150 μl of the BAY 11-7082 clinical trial previous reaction by Combretastatin A4 addition of 150 μl of a glucose oxidase/peroxidase mixture (0.66 mg ml-1) Aspergillus niger glucose oxidase and 0.25 mg ml-1 horseradish peroxidase in 0.5 M phosphate buffer, pH 6.0 (Sigma) and 50 μl of 2.33 mg ml-1 o-toluidine. After 30 min incubation at 37°C, 1.5 ml of water was added to the samples and absorption was measured at 420 nm in a Perkin Elmer Lambda 25 UV/Vis spectrophotometer and compared to glucose standards in a concentration range of 0-300 μg ml-1. Finally, trehalose content was inferred from the glucose content by performing a standard curve with commercial trehalose (Sigma) ranging from 1 to 5 mM. Trehalose concentration was expressed as μmol mg Mirabegron protein-1. Isolation of the otsA and 16S rRNA genes Total DNA was isolated by using the CTAB method [53]. Amplification of about 1-kb of the otsA gene from R. gallicum bv. phaseoli 8a3, R. leguminosarum bv. phaseoli 31c3, and R. etli 12a3 was performed by

using the primers OTA1: 5′-ATC TGG ATG GGA TGG TCG GGA-3′ and OTA2: 5′-GAC ATA TTC CTT GGC AAC GAG GTT-3′. For strain CIAT 899, otsA was amplified by using the degenerated primers: OTAS1: 5′-CAT CTG GAT GGG (CT)TG GTC GG-3′ and OTAS2: 5′-GGC GAC ATA TTC CTT GGC (GC)AC (GC)AG GTT-3′. The amplification protocol consisted of the following steps: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation (45 seconds at 94°C), annealing (45 seconds at 58°C), extension (1 min at 72°C), and a final extension step at 72°C for 10 min. Sequencing of the otsA genes was performed by the company Newbiotechnics (NBT, Seville, Spain). PCR amplifications of the complete 16S rRNA genes were carried out as previously described [54].

The time we had during our project was enough to develop an approach to identify the different issues to be included in a monitoring system (i.e. time, seasonal calendar, people’s availability, necessity of a multi-stakeholder engagement, selection of simple but important

NTFPs). The repetition of assessments and measurements, and data quality control needs regular visits to the monitored villages. In our case, the 2 year-duration of our research was not enough to achieve long-term impacts. It did not allow real testing. We were only able to test the monitoring system for 6 months, which did not cover a full season of NTFP collection. Unpredictable events were among the limitations we identified for full implementation of the monitoring system. We recommend at least two cycles of NTFP harvest (i.e. 2 years), which would allow comparison, to test the approach and learn from the results. Integration into eFT508 molecular weight national policies (here PLUP)

was in progress at the end of the project (Lestrelin et al. 2011, Bourgoin and Castella 2011, Bourgoin et al. 2012), but we lacked time to discuss with decision-makers ways the monitoring could be used to SC79 clinical trial assess the impact of LUP and to scale up. Scaling up The monitoring system developed in Laos has the potential to address multi-stakeholders’ concerns: villagers, including local elites, local authorities at the kumban and district levels, and organizations working on community development and conservation. Integrating these management practices into multi-level and multi-scale governance could support win-win solutions for both the villagers (data to negotiate) and the district authorities (data to deliver to the provincial level). If embedded in existing local governance and applied in key government policies, it could

Fludarabine be used as a tool to empower local communities. This could be achieved by providing them with information on the effects of land management policies on forest resources and livelihoods. The different steps we propose could be applied easily to different situations elsewhere in the country. This could be with different ethnic groups, involving villages at different steps of rural transition, and different scales, from the village level to the village cluster and to the landscape. For the time being, we can only share the potential of this approach and call for more implementation trials before expanding it to different situations and provinces in the country. Acknowledgments The authors thank the www.selleckchem.com/products/LY294002.html Viengkham community for their participation to their activities. They also thank Glen Mulcahy, Douglas Sheil and the anonymous reviewer for their valuable comments and editing, and Mohammad Agus Salim for designing the maps. They acknowledge the Swiss Agency for Development and Cooperation (SDC) and the European Commission for their financial support.

These observations match earlier data that described detectable levels of metabolism of NeuNAc in most oral streptococci, while sialidase activity could only

be found in few species [32]. Amongst the oral streptococci, pneumococci carry a composite locus, probably assembled from the gene pool of related species. The association of the SPG1594 oxidoreductase with ManNAc metabolism and of two small hypothetical proteins (SPG1586 and SPG1588) with NeuNAc metabolism remains RepSox clinical trial unexplained, as all necessary enzymes for sialic acid metabolism appear to be already present. The PTS transporter, found to transport glucosamine, appears to be unique in pneumococci [23]. The fact that glucosamine is the last metabolic intermediate in sialic acid catabolism may indicate a convenience for the bacterium in co-utilisation of GlcN and ManNAc, even if it is not clear where pneumococci Selleckchem AZD5363 should feed on GlcN, a rare sugar in the human nasopharynx, but of which on the contrary the pneumococcal cell wall is exceptionally

rich [33]. When pneumococci grow on ManNAc and NeuNAc as the sole carbon sources, the generation time is much longer than on glucose or on the yeast-extract derived carbohydrates of the CAT medium, which is in accordance with previous data [23]. Growth on ManNAc (Figure 3B, Figure 4A) shows a profile with a change in generation time. In the case of growth on glucose repression of the whole locus indicates sequential

utilisation of sugars. This is less selleck screening library clear for the growth on yeast extract derived dextran and ManNAc, where only part of the locus is induced with the exception of the predicted central transcriptional unit encoding the principal ManNAc ABC transporter SPG1596-8. The data here presented thus do not rule out, that during growth on yeast derived sugars also ManNAc may be co-metabolised. The differential impact of regulation on the three operons Protirelin is reminiscent of data on expression of this locus in transparent colony variants, where also the nanB and ManNAc-uptake operon is not involved in differential expression, while the other two transcripts are upregulated [21]. The fact that both ManNAc and NeuNAc are able to efficiently induce the operon is in accordance with our finding that the SPG1583 regulator acts a positive regulator, as documented by absence of metabolism in its mutant and also by its annotation as a phosphor-sugar binding regulator. Since NeuNAc is imported by an ABC transporter, which does not phosphorylate during uptake, and is first hydrolysed to ManNAc before becoming phosphorylated (Figure 1B), both amino sugars may equally originate the inducer of the positive regulator; probably ManNAc-phosphate.

J Clin Microbiol 2005,43(1):66–73.PubMedCrossRef 29. Johnson JR, Owens KL, Clabots CR, Weissman SJ, Cannon SB: Phylogenetic relationships among clonal groups of extraintestinal pathogenic Escherichia coli as assessed by multi-locus sequence analysis. Microbes and infection /Institut Pasteur CYT387 purchase 2006,8(7):1702–1713.PubMedCrossRef 30. Moulin-Schouleur M, Schouler C, Tailliez P, Kao MR, Bree A, Germon P, Oswald E, Mainil J, Blanco M, Blanco J: Common virulence factors and genetic relationships between O18:K1:H7 Escherichia coli isolates of human and avian origin. J Clin Microbiol 2006,44(10):3484–3492.PubMedCrossRef 31. Levy SB,

FitzGerald GB, Macone AB: Spread of antibiotic-resistant click here plasmids from chicken to chicken and from chicken to man. Nature 1976,260(5546):40–42.PubMedCrossRef 32. Linton AH, Howe K, Bennett PM, Richmond MH, Whiteside EJ: The colonization of the human

gut by antibiotic resistant Escherichia coli from chickens. J Appl Bacteriol 1977,43(3):465–469.PubMedCrossRef 33. Ojeniyi AA: Direct transmission of Escherichia coli from poultry to humans. Epidemiol Infect 1989,103(3):513–522.PubMedCrossRef 34. van den Bogaard AE, Willems R, London N, Top J, Stobberingh EE: Antibiotic resistance of faecal STI571 research buy enterococci in poultry, poultry farmers and poultry slaughterers. J Antimicrob Chemother 2002,49(3):497–505.PubMedCrossRef 35. Moulin-Schouleur M, Reperant M, Laurent S, Bree A, Mignon-Grasteau Niclosamide S, Germon P, Rasschaert D, Schouler C:

Extraintestinal pathogenic Escherichia coli strains of avian and human origin: link between phylogenetic relationships and common virulence patterns. J Clin Microbiol 2007,45(10):3366–3376.PubMedCrossRef 36. Hagan EC, Mobley HL: Haem acquisition is facilitated by a novel receptor Hma and required by uropathogenic Escherichia coli for kidney infection. Mol Microbiology 2009,71(1):79–91.CrossRef 37. Bonacorsi SP, Clermont O, Tinsley C, Le Gall I, Beaudoin JC, Elion J, Nassif X, Bingen E: Identification of regions of the Escherichia coli chromosome specific for neonatal meningitis-associated strains. Infect Immun 2000,68(4):2096–2101.PubMedCrossRef 38. Dozois CM, Daigle F, Curtiss R: Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc Natl Acad Sci U S A 2003,100(1):247–252.PubMedCrossRef 39. Feldmann F, Sorsa LJ, Hildinger K, Schubert S: The salmochelin siderophore receptor IroN contributes to invasion of urothelial cells by extraintestinal pathogenic Escherichia coli in vitro. Infect Immun 2007,75(6):3183–3187.PubMedCrossRef 40. Peigne C, Bidet P, Mahjoub-Messai F, Plainvert C, Barbe V, Medigue C, Frapy E, Nassif X, Denamur E, Bingen E, Bonacorsi S: The plasmid of Escherichia coli strain S88 (O45:K1:H7) that causes neonatal meningitis is closely related to avian pathogenic E.

​ncbi.​nlm.​nih.​gov/​ revealed that the components of this efflux system shared amino acid sequence identity with the well characterized AcrAB-TolC, BpeAB-OprB, and MexAB-OprM RND efflux pumps of E. coli, B. pseudomallei, and P. aeruginosa, respectively. In particular, BCAS0592 shared 60, 59, 56% amino acid identity with the RND transporters AcrB (E. coli), BpeB (B. pseudomallei), and MexB (P. aeruginosa), respectively. BCAS0591 shared 53, 50, and 50% amino acid identity with the membrane fusion proteins AcrA (E. coli), MexA (P. aeruginosa), and BpeA (B. pseudomallei). On the other hand, BCAS0593 shared selleck products 52% amino acid identity

with OprM (P. aeruginosa) and 49% with OprB (B. pseudomallei), both of which are outer membrane pore proteins. Figure 1 Genetic map of B. cenocepacia rnd operons containing the BCAS0592, BCAL1675, and NVP-AUY922 BCAL2821 genes. Gene positions and orientations are shown. Membrane fusion protein encoding genes are depicted in green, the RND encoding Napabucasin ones in yellow (the previous name attributed to these genes in reported in parentheses), and the genes encoding outer membrane

proteins are in white. The putative repressor gene BCAL1672 is depicted in pink. The operon encoding RND-3 is located on chromosome 1 and spans nucleotides 1830038 to 1834638. The first gene, BCAL1674, encodes the membrane fusion protein, a predicted 406-aa protein. The product of the downstream gene is a predicted 1046-aa protein that functions as an RND transporter. The third gene, BCAL1676, encodes the 486-aa outer membrane pore protein [Fig. Suplatast tosilate 1]. BLASTP results revealed that BCAL1674 had 79 and 48% identity with the membrane fusion proteins AmrA (B. pseudomallei) and MexC (P. aeruginosa), while BCAL1675 was similar to AmrB (B. pseudomallei, 86%) and to MexD of P. aeruginosa (52%), both encoding the RND transporter. BCAL1676 was highly related to the outer membrane proteins OprA of B. pseudomallei (78% of identity) and OprM of P. aeruginosa (47%) and again possessed the predicted conserved structural features of outer membrane proteins that function in RND efflux systems.

A gene encoding a predicted TetR family regulator protein (BCAL1672) is located upstream of BCAL1674 but is transcribed in the opposite direction [Fig. 1]. Lastly, the predicted operon encoding RND-4, comprising the genes BCAL2820, BCAL2821 and BCAL2822, is located on chromosome 1 and spans nucleotides 3095788 to 3101801 [Fig. 1]. BCAL2821 encodes the 1066-aa RND transporter protein, which is highly related to BpeB from B. pseudomallei (94% identity) and to MexB (P. aeruginosa, 64% identity). BCAL2820 encodes the 507-aa outer membrane protein related to OprB (B. pseudomallei, 84% identity) and to OprM from P. aeruginosa (53% identity). BCAL2822 encodes a predicted 424-aa membrane fusion protein highly similar to BpeA from B. pseudomallei (89% identity) and to MexA from P. aeruginosa (54% identity).

Table 1 Functions over-represented in A. vulgare ovaries in response to Wolbachia infection.   Biological process GO accession A S A/S AO ~ SO cell fate determination GO:0001709 0.02 0.05 0.40 level 3 immune effector process GO:0002252 0.07 0.16 0.44 (n= 99) regulation of immune system process GO:0002682 0.04 0.14 0.29   generation of a signal involved in cell-cell signaling GO:0003001 0.04 0.05 0.80   Apoptosis inhibitor muscle contraction

GO:0006936 0.02 0.07 0.29   chromosome segregation GO:0007059 0.18 0.23 TPCA-1 0.78   ensheathment of neurons GO:0007272 0.00 0.02 0.00   circadian rhythm GO:0007623 0.07 0.09 0.78   cell recognition GO:0008037 0.02 0.07 0.29   reproductive behavior GO:0019098 0.04 0.05 0.80   membrane docking GO:0022406 0.04 0.05 0.80  

viral reproductive process GO:0022415 0.02 0.05 0.40   cellular pigmentation GO:0033059 0.04 0.05 0.80   leukocyte activation GO:0045321 0.05 0.09 0.56   regulation of response to stimulus GO:0048583 0.12 0.18 0.67   coagulation GO:0050817 0.09 0.11 0.82   regulation of body fluid levels GO:0050878 0.04 0.05 0.80   endocrine process GO:0050886 0.11 0.14 0.79   cellular response to stimulus GO:0051716 0.05 0.07 0.71 In the same manner, two in vitro SSHs were performed by subtracting common transcripts between symbiotic and asymbiotic ovaries (SSH-S), and reciprocally (SSH-A). These selleck kinase inhibitor SSHs were contaminated by a high proportion of mitochondrial ESTs (~40%) that were removed for further analyses. To reveal the functions over-represented, we compared each SSH to SO library by the FatiGO web tool. One biological process (vesicle transport along microtubule) and one molecular function (microtubule motor activity) were over-represented in asymbiotic ovaries (Table 2). Most of the 223 unigenes that are associated to these two

GO terms belong to the kinesin-like protein family. In these two libraries, the BLAST analyses allowed the identification of 1 immune gene in SSH-S and 6 immune Casein kinase 1 genes in SSH-A libraries respectively (Additional File 4: Immune unigenes present in SO, AO, SSH-S, SSH-A, SSH-C, and SSH-NC libraries). Table 2 Functional enrichment analysis: list of GO terms that were over-represented in the lists of unigenes obtained by SSH experiments on ovaries (FatiGO web tool). P-value of Fisher’s exact unilateral tests. Adjusted p-value for multiple test correction. Test # unigenes Ontology domain Level Term GO ID p-value Adj. p-value SSH-A versus SO 223 Biological process 9 vesicle transport along microtubule GO:0047496 1.35E-04 5.97E-02     Molecular function 3 microtubule motor activity GO:0003777 1.13E-03 9.85E-02 SSH-S versus SO 44     no significant term       In order to identify genes expressed in response to pathogenic bacteria, we performed SSH libraries between S. typhimurium-challenged and unchallenged asymbiotic A. vulgare females (SSH-C) and reciprocally (SSH-NC).

References 1. Stitch SR, Toumba JK, Groen MB, Funke CW, Leemhuis J, Vink J, Woods GF: Excretion, isolation and structure of a new phenolic constituent of female urine. Nature 1980,287(5784):738–740.PubMedCrossRef 2. Setchell KD, Lawson AM, Mitchell FL, Adlercreutz H, Kirk DN, Axelson M: Lignans in man and in animal

species. Nature 1980,287(5784):740–742.PubMedCrossRef 3. Wang LQ: Mammalian phytoestrogens: enterodiol and enterolactone. Angiogenesis inhibitor Journal of chromatography 2002,777(1–2):289–309.PubMedCrossRef 4. Adlercreutz H, Mousavi Y, Clark J, Hockerstedt K, Hamalainen E, Wahala K, Makela T, Hase T: Dietary phytoestrogens and cancer: in vitro and in vivo studies. The Journal of steroid biochemistry and molecular biology 1992,41(3–8):331–337.PubMedCrossRef 5. Kitts DD, Yuan YV, Wijewickreme AN, Thompson LU: Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone. Molecular and cellular biochemistry

1999,202(1–2):91–100.PubMedCrossRef 6. Lemay A, Dodin S, Kadri N, Jacques H, Forest JC: Flaxseed dietary supplement versus hormone replacement therapy in hypercholesterolemic menopausal women. Obstetrics and gynecology 2002,100(3):495–504.PubMedCrossRef 7. Adlercreutz H: Lignans and human health. Critical reviews in clinical laboratory Tanespimycin sciences 2007,44(5–6):483–525.PubMedCrossRef 8. Thompson LU, Robb P, Serraino M, Cheung Apoptosis inhibitor F: Mammalian lignan production SPTLC1 from various foods. Nutrition and cancer 1991,16(1):43–52.PubMedCrossRef

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Therefore, CHLA and PUG are able to abrogate host cell binding and penetration by HCMV, HCV, DENV-2, MV, and RSV during the cell entry process. Control of virus spread post-infection by CHLA and PUG We next determined the

antiviral activity of the two hydrolyzable tannins in controlling spread of established infections. Target cell monolayers were infected with the respective test virus, and then incubated with or without the compounds. As shown in Figure 6, both CHLA and PUG effectively inhibited FHPI chemical structure HCMV, HCV, and MV infections (80 – 100% protection), but were ineffective against the growth of DENV-2 and RSV (< 25%). To further validate the tannins’ effect on virus cell-to-cell transmission, we examined the effects of the drugs on viral plaque size. The change in the area of the Buparlisib cell line plaques was measured using either viral immunofluorescence or EGFP-tagged reporter viruses. Neutralizing antibodies, methylcellulose or KU55933 solubility dmso agarose were included in the overlay medium to prevent secondary infection of uninfected cells throughout the monolayer, ensuring that viral spread occurs

via intercellular junctions between neighboring infected and virus-free populations. The data indicated

that viral plaques from HCMV, HCV, and MV infections were restricted by CHLA and PUG to near initial size, whereas plaques due to DENV-2 and RSV infections were unaffected and expanded further (Figure 7 and Additional file 1: Figure S1, Additional file 2: Figure S2, Additional click here file 3: Figure S3, Additional file 4: Figure S4 and Additional file 5: Figure S5). These results are in agreement with the data obtained following post-entry drug treatment in Figure 6, where HCMV, HCV, and MV, but not DENV-2 and RSV, were shown to be sensitive to the tannins’ antiviral effects. Thus, it appears that the two tannins are effective in limiting post-infection spread of HCMV, HCV, and MV, but are inefficient in preventing cell-to-cell transmission of DENV-2 and RSV. Heparin, on the other hand, displayed limited effect against the spread of the viruses post-entry (Figures 6 and 7). The window of antiviral activity from CHLA, PUG, and heparin at different stages of viral entry and spread are summarized in Table 3.

Protein concentrations of the supernatant (cytosolic fraction) were measured using the colorimetric assay RC DC Protein Assay (Bio-Rad), using bovine serum albumin (BSA) as standard

protein, according to the manufacturer’s instructions. The supernatants were stored in aliquots at -80°C. Two-dimensional gel electrophoresis conditions Aliquots of the L. sakei cytosolic fraction corresponding to 50 μg (analytical gel) or 200 μg (preparative gel) of protein were diluted by adding a rehydration buffer (6 M urea (Merck), 2 M thiourea (Merck), 4% 3- [(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich), selleck chemicals llc 0.5% immobilized pH gradient (IPG) buffer pH 4-7 (GE Healthcare Bio-Sciences), and 2.5% dithiothreitol (DTT; Bio-Rad)) to a final volume of 380 μl. This solution was

used to rehydrate 18-cm pH 4-7 linear IPG strips (GE Healthcare BioSciences). Strips were passively rehydrated at room temperature for 12-16 h under mineral oil, before isoelectric focusing (IEF) was performed in an Ettan IPGphor II unit (GE Healthcare Bio-Sciences, Uppsala, Sweeden) as follows: 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, from 1000 to 8000 V in 30 min, and finally 8000 V for 6 h. CP-868596 purchase The strips were incubated at room temperature for 15 min in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/l) glycerol (Merck) and 2% (w/v) sodium dodecyl sulfate (SDS; Shelton Scientific)) supplemented with 1% (w/v) DTT, followed by 15 min in equilibration buffer containing 2.5% (w/v) iodoacetamide (Merck). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide gels was carried out with an Ettan DALT II system (GE Healthcare Bio-Sciences, Uppsala, Sweeden). Proteins were resolved at 20°C at a current of 2.5 mA/gel for 45 min and then at 25 mA/gel until the tracking dye had migrated to the bottom of the gel. Analytical gels were silver stained as described by Blum et al. [37] and preparative gels according to Shevchenko et al. [38]. For the final analysis, three 2-DE see more gels were

run from each Microbiology inhibitor strain from each of the two independent bacterial cultures. Image and statistical analysis Digitized 2-DE images (16-bit greyscale, 300 dpi) of the stained gels were acquired with an office scanner (Epson Perfection 4990 Photo, Epson) and imported into Progenesis SameSpots software v.3.1 (Nonlinear Dynamics). For each strain, five glucose images and five ribose images were aligned using one selected glucose image as a reference [39]. Spots were detected simultaneously across the images leading to one spot map, an approach which addresses the problems of missing values and reduces variance in spot volume across biological or technical replicates by applying the same spot outline across the image series [39, 40]. The spot pattern was manually edited, gel artefacts were removed, and images were grouped glucose vs. ribose.

The reactive proteins were visualized using ECL-plus (Amersham) according to the manufacturer’s instructions. As an internal standard, anti-βselleck screening library -actin mouse monoclonal antibody (Sigma) was used as the primary antibody to detect β-actin protein. In vitro migration and click here invasion assays Migration was analyzed in a Boyden chamber assay using Falcon cell culture inserts (pore size, 8.0 μm; Becton Dickinson, Franklin Lakes, NJ, USA). Analysis of invasive properties was achieved by using Falcon cell culture inserts covered with 50 μg of Matrigel (Becton Dickinson). For both assays, the upper chamber of the insert was filled with 500 μL of the cell and drug suspension (5×103 cells) and conditioned medium (addition of RANKL in

serum-free medium) was added to the lower chamber. After the cells had been incubated for 24 hr, the remaining cells in the upper layer were swabbed with cotton and penetrating cells in the lower layer were fixed with 95% ethanol and removed for hematoxylin staining. Cells passing through the 8 μm-pore culture inserts were counted using light microscopy. Statistical analysis All results are

expressed as means and S.D. of several independent experiments. Multiple comparisons of the data were done by ANOVA with Dunnet’s test. P values less than 5% were regarded as significant. Results RANKL promotes the EMT, migration, and invasion of breast cancer cells and normal mammary epithelial cells In order to determine the induction of EMT by RANKL in breast cancer cells, we investigated the change Wortmannin price in morphology following stimulation

with RANKL. After 48 h of treatment, the morphology of 4T1, MCF-7, and NMuMG cells changed from an epithelial sheet-like structure to a mesenchymal fibroblastic spindle shape, which is characteristic of EMT (Figure 1A). We also found that these cells expressed RANK else (data not shown). Next, in order to investigate the molecular mechanism of RANKL-mediated EMT of breast cancer cells and normal mammary epithelial cells, we examined the effects of RANKL on EMT markers. RANKL stimulation resulted in downregulation of the mRNA of the epithelial marker E-cadherin and upregulation of the mRNAs of the mesenchymal markers vimentin and N-cadherin in a concentration-dependent manner in 4T1, MCF-7, and NMuMG cells (Figure 1B–1D). The expression levels of the transcriptional repressors of E-cadherin, Snail and Twist, were upregulated by RANKL treatment in 4T1, MCF-7, and NMuMG cells (Figure 1B–1D). However, no significant change in the level of Slug mRNA was detected in RANKL-treated cells as compared to control cells in 4T1, MCF-7, and NMuMG cells (phosphate-buffered saline-treated cells) (Figure 1E–1G). In addition, small interfering RNA-mediated silencing of RANK expression suppressed RANKL-induced upregulation of vimentin, N-cadherin, Snail, and Twist mRNAs and RANKL-mediated downregulation of E-cadherin mRNA (data not shown).