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pylori and L acidophilus determined by the percentage of LDH

pylori and L. acidophilus determined by the percentage of LDH leakage (in triplicate) and non-stained trypan blue (see more single) Bacteria and MOI Cytotoxicitya (% LDH) Viable cell count (× 106) Cell only for 4 and 8 hours 18.0, 18.0

1.36 H. pylori for 4 hours     MOI 100 18.1 selleck inhibitor 1.00 Lactobacillus for 8 hours     MOI 1 18.4 1.00 MOI 10 18.0 1.11 MOI 100 18.7 1.24 MOI 1000 24.2 0.77 aAll cytotoxicity data were presented with mean value of three tests H. pylori stimulated IL-8 and TNF-α but not TGF-β1 production in vitro In MKN45 cells incubated with H. pylori (MOI 100) at various time periods, the IL-8 level increased from the 4th to the 8th hour after co-incubation, as determined by ELISA (Figure 1A). For TNF-α, the post-incubation level rose after the 4th hour and maintained a plateau until the 8th hour (Figure 1B). However, the TGF-β1 level did not increase after H. pylori incubation for 4 hours (data not shown). Figure 1 (A) IL-8 and (B) TNF-α concentrations in the supernatant of MKN45 cells culture after variable duration of H. pylori and L. acidophilus

infection (MOI = 100). Data were expressed as means ± standard deviation (SD) (in triplicate). this website In contrast, L. acidophilus did not induce IL-8, TNF-α, and TGF-β1 expressions of MKN45 at least within the 8-hour co-incubation period. Pre-treatment of L. acidophilus attenuated H. pylori-induced IL-8 Because the IL-8 level of MKN45 cells could be induced by H. pylori challenge for 4 hours, the time- and dose-dependent effects of probiotics in reducing pro-inflammatory cytokines and TGF-β1 on the 4th hour were

studied. The IL-8 and TGF-β1 concentrations were Thymidylate synthase shown for MKN cells challenged by H. pylori and with variable doses of L. acidophilus pretreatment for 8 hours (Figure 2). Compared to the control group, L. acidophilus pre-treatment with higher bacterial colony count (MOI 100) reduced H. pylori-induced IL-8 expressions in MKN45 cells (P < 0.05). The TGF-β1 level did not change (P > 0.05). Figure 2 The concentrations of IL-8 (blank column) and TGF-β1 (black column) in the supernatant of MKN45 cells pre-treated with different MOI (0: control; 1: 1 × 10 6 c.f.u.; 10: 1 × 10 7 c.f.u.; 100: 1 × 10 8 c.f.u.) of L. acidophilus. The cells were washed thrice with PBS to remove the L. acidophilus and then infected with H. pylori (MOI = 100) for 4 hours. Data are expressed as means ± SD (in triplicate). Statistical analysis was performed in each measurement with comparisons to the controls (cells treated H. pylori only; IL-8 2034 ± 865 pg/ml and TGF-β1 587.2 ± 39.8 pg/ml) (*P < 0.05). L. acidophilus reduced H. pylori-induced NF-κB by increasing IκBα The study determined that MKN45 cells (MOI 100) incubated with H. pylori led to a peak increase of nuclear NF-κB production within one hour. Thus, nuclear NF-κB levels of MKN45 cells co-incubated with H. pylori, after prior pre-treatments by various MOIs (1-100) of L.


Absence RSL3 price of DNA was verified by qPCR. Each RNA sample (300 ng) was reverse transcribed using random hexamer oligonucleotides (Bioline, London, UK). Specific primers were designed to amplify an approximately 100 bp region of each gene in the study (Additional file 1: Table S1). qPCR was performed using a StepOnePlus™ Real-Time PCR System (Applied buy Barasertib Biosystems); each reaction consisted of 1 μl of cDNA, 1 x SensiMixPlus SYBR (Quantace, London, U.K.), 200 nM of specific primers

in a 25 μl reaction. The amplification cycling conditions were: initial denaturation at 95°C for 10 min; 39 cycles of denaturation at 95°C for 10 s; annealing at 60°C for 30 s; extension at 72°C for 5 s. A melting curve analysis was performed for each amplification reaction, with a temperature gradient of 0.1°C from 55°C to 95°C. No-template controls and a calibration curve, consisting of 6 dilutions of the selleck chemicals llc PCR amplicon of each gene cloned into PCR-Blunt vector (Invitrogen, Paisley,

U.K.) linearised with Nco I (NEB, Herts, U.K.), were included in every experiment (Additional file 1: Table S1). Statistical analysis was performed using a one-way ANOVA comparing gene copy numbers at different time points in each experiment to test the hypothesis that there is no variation in gene copy number during the recovery period. A post-hoc Dunnett’s test was employed, using the sample corresponding to the lysogen culture (-60) as the reference group, to assess whether or not time points

differed from the reference. P < 0.05 values were considered to be statistically significant. Protein extraction for 2D-PAGE Cultures of MC1061 and MC1061(Φ24B) were incubated for 6 h at 37°C. Cells were harvested and pellets washed in 1 ml of wash solution PIK3C2G (10 mM Tris-HCl, pH 8.0; 1.5 mM KH2PO4; 68 mM NaCl; 9 mM NaH2PO4). Cells were resuspended in 1 ml of resuspension buffer (10 mM Tris-HCl, pH 8.0; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM DTT; 0.1% SDS; 20 μl of protease inhibitor [Roche CompleteMini EDTA Free protease inhibitor cocktail tablets]) and each sample was sonicated for 5 × 10 s. DNase was added (5 μg ml-1) and samples were incubated for 1 h at 37°C. Samples were centrifuged for 1 h at 12,000 g, the supernatant recovered and protein concentration determined using the Bradford Assay. Aliquots (110 μg protein) of the sample were taken and precipitated in 10% TCA in acetone containing 20 mM DTT for 45 minutes at -20°C. Pellets were washed twice in ether. 2D-PAGE Isoelectric focussing was carried out on 18 cm IPG strips (pH 4-7,3-5.6 and 5.3-6.5;GE Healthcare), at 3,500 V for 7 h. Proteins were separated in the second dimension on 1.5 mm 4% stacking/15% resolving SDS-PAGE gels, for 6.5 h at 20 W per gel (up to maximum of 180 W). Proteins were silver stained [50].

Tumor-infiltrating cells in control, un-disturbed tumors were ran

Tumor-infiltrating cells in control, un-disturbed tumors were randomly located and no specific distribution pattern can be identified. In irradiated tumors, except the aggregation of CD68 positive macrophages at chronic hypoxia region, we further found that CD11b and Gr-1 positive cells were concentrated in central necrotic region and F4/80 positive macrophages were distributed along the junction of necrotic and chronic hypoxic region. Flow cytometry assay

demonstrated that total CD11b cells were not altered, but there are more CD11b and Gr-1 positive cells in the necrotic region of irradiated tumor than control tumor, no matter the size of tumor or necrotic area. The re-distribution pattern of different subsets of CD11b positive cells into different microenvironments in irradiated tumors suggest DNA Damage inhibitor irradiated tumors form sub-component

which has factor(s) to attract specific subset of CD11b positive cells. The illustration of the role and function of these cells in particular regions may provide a new strategy to improve the effectiveness of radiation therapy. (This work is supported by grants of NHRI-EX98-9827BI and NTHU-98N2425E1 to Chi-Shiun Chiang) Poster No. 212 Single-Chain Akt tumor Antibodies against GW2580 ic50 the HGF/SF Receptor Danielle DiCara 1,3 , Zhe Sun2, John McCafferty2, Ermanno Gherardi1 1 Growth Factors Group, MRC Centre, Cambridge, UK, 2 Department of Biochemistry, University of Cambridge, Cambridge, UK, 3 Department of Oncology, University of Cambridge, Cambridge, UK Dysregulation of the Met receptor tyrosine kinase and of its cognate

ligand Hepatocyte Growth Factor / Scatter Factor (HGF/SF) occurs frequently in cancer, and Met overexpression indicates poor prognosis in several cancers such as breast and head and neck. HGF/SF Miconazole binding triggers signalling that promotes cancer cell migration, proliferation and invasion. We have generated Met-binding single-chain fragment variable (scFv) antibodies by phage display, using the ‘McCafferty’ library, which has a diversity of 1010 clones. After two rounds of biopanning, 76/182 clones bound Met in ELISA, of which 72 were found to be unique. Preliminary data indicates isolation of several clones capable of inhibiting HGF/SF-induced scatter of the pancreatic cancer line BxPC-3. Affinity maturation and selection strategies directed towards antibodies that bind the same epitopes as HGF/SF may yield clones with higher activity. Met-blocking scFv may be useful for cancer therapy. This work is funded by Cancer Research UK / Cancer Research Technology. Poster No.

aureus 43300(106 CFU/ml) intranasally Group 2: Mice were administ

aureus 43300(106 CFU/ml) intranasally Group 2: Mice were administered S. aureus 43300, left for a period of 48 hours to allow

nasal colonisation followed by intranasal administration of selleck chemical 50 μl of phage (107 PFU/ml) given twice (at an interval of 24 hours). Group 3: Mice were administered S. aureus 43300, left for a period of 48 hours to allow nasal colonisation followed by intranasal administration of 50 μl of mupirocin (5 mg/kg dissolved in water; given once) the next day. Group 4: Mice were administered S. aureus 43300, left for a period of 48 hours to allow nasal colonisation followed by intranasal administration of phage as well as mupirocin (5 mg/kg) the next day. The parameters used to monitor colonization included a) Bacterial load (CFU/ml) in nares b) Phage counts in nares c) Nasal myeloperoxidase (MPO) levels and e) Histopathological examination Nasal bacterial OSI-027 research buy load Four mice from each of group were taken and sacrificed on day 2, 5, 7, 10, 12 post treatment by selleck chemicals cervical dislocation. The nasal region was wiped

externally with 70% ethanol, nose was removed along with nasal bone. The entire nasal tissue was excised using sterile scissors and homogenized. The homogenates were plated quantitatively on nutrient agar containing 20 μg/ml of ampicillin to select S. aureus 43300 after overnight incubation at 37°C. Nasal homogenates were also processed to determine the phage titer by modified double layer agar method [20]. Myeloperoxidase (MPO) estimation Mice from each group (same groups as those categorized for phage protection studies with 20 animals per group) were killed

and their nasal tissue was excised and homogenised in 50 mM PBS (pH 7.4). Nasal samples were processed for MPO determination as per the method of Greenberger et al. [21]. The absorbance was read immediately at 490 nm over a period of 4 minutes. MPO was calculated as the change in optical density (O.D) x dilution factor (D.F). Histopathological examination Extent of injury caused by S. aureus and healing of the colonized mouse nose following therapy with phage or antibiotic was assessed on the basis of histopathological analysis of the injured and recovered nose according to the method of Brans et al. [22]. The sections were picked Digestive enzyme on separate slides, stained with hematoxylin and eosin (Hi-Media, Mumbai) and the slides then examined under a microscope to evaluate the extent of damage. Statistical methods The data is expressed as mean ± standard deviation of replicated values where indicated. The statistical significance of differences between groups was determined by Student’s t-test (two groups),one-way ANOVA followed by a Tukey test using Sigma Stat, Graph pad prism (Graph pad software, San Diego, CA). p value of less than 0.05 and 0.01 was considered statistically significant for a confidence interval of 95% and 99% respectively. Results The nasal epithelial cells were isolated from mouse nasal tissue and cultured at 37°C in presence of 5% CO2.


Figure find more 4 Expression profiles of five known genes of T. harzianum determined by Northern blot hybridization. The fungus was cultured in MS basal medium alone

or in the presence of tomato plants (MS-P), 2% glucose (MS-G), or 1% chitin (MS-Ch), as described in Methods. Fungal 18S rDNA was used as a loading control. Identification of T. harzianum genes expressed in response to tomato plants Since we were interested in identifying the genes induced in T. harzianum CECT 2413 by the presence of tomato plants, we selected the 257 probe sets affording significant differential expression in MS-P vs. MS (fold-change greater than 2.0 and FDR = 0.23; see additional file 3), and the corresponding transcript sequences were annotated according to the GO classification and the hierarchical structure using the Blast2GO suite [27]. GO categories were assigned to 85 of the 257 sequences examined (see additional file 4) whereas another 57 had no results after mapping or annotation processes (many of them were hypothetical proteins), and the remaining 115 sequences did not yield significant hits in the databases. As summarized in additional file 5, the annotated sequences represented a total of 46 different genes. Additionally, three sequences without Blast2GO annotation (T34C26, T34C242 and L10T34P112R10010)

but corresponding to three portions of the known protein QID74 [Prot: O74567] of T. harzianum CECT 2413 were also included in additional file 5. Within the genes identified as showing up-regulation in MS-P vs. MS, about 45% were

genes encoding homologues of proteins involved in metabolic pathways, mainly enzymes for carbohydrate, Doramapimod in vivo lipid and amino acid metabolism, but also enzymes for vitamin and cofactor biosynthesis, and energy- Rebamipide and detoxification- related processes. Interestingly, some of these up-regulated genes (encoding O-glycosyl hydrolase family 2, aldose 1-epimerase, dihydroxyacetone kinase, acid sphingomyelin phosphodiesterase, GTP cyclohydrolase I, glutathione-dependent formaldehyde-activating enzyme, plus two hypothetical proteins) were classified according to Blast2GO in the functional category “”growth or development of symbiont on or near host surface”" since their homologues in Magnaporte grisea were differentially expressed during appresorium formation [28]. Proteins related to carbohydrate metabolism included several enzymes of the glycolysis/gluconeogenesis pathways plus a phosphoketolase of the pentose phosphate pathway, and a 1,3-beta-glucan synthase involved in cell wall biosynthesis. The three up-regulated genes with homologues in lipid metabolism corresponded to a phosphatidylserine synthase participating in phospholipid biosynthesis; a dihydroxyacetone kinase involved in glycerolipid metabolism, and an acid sphingomyelin phosphodiesterase, responsible for breaking sphingomyelin down into phosphocholine and ceramide.

CSE1L was recently shown to associate with a subset of p53 target

CSE1L was recently shown to associate with a subset of p53 target promoters, and reduced CSE1L expression decreased 53-mediated transcription and thus lowered apoptosis [31]. Our studies

showed that increased CSE1L expression can enhance doxorubicin-induced p53 accumulation [12, 13]; therefore, CSE1L regulates p53 protein accumulation induced by chemotherapeutic drugs. Other studies of ours also showed that interferon-γ treatment increased CSE1L expression in cancer cells [23] and interferon-γ co-treatment enhanced doxorubicin-induced selleck inhibitor p53 accumulation of Hep G2 hepatoma cells [32]. Thus, interferon-γ may increase doxorubicin-induced p53 accumulation by modulating CSE1L expression. CSE1L is highly expressed in cancer, and FG4592 the results of our studies suggest that CSE1L plays a role in regulating p53 accumulation induced

by chemotherapeutic drugs. Therefore, CSE1L may play an important role in mediating the cytotoxicities of chemotherapeutic drugs against cancer cells in cancer chemotherapy. Also, CSE1L may be a target for developing strategies to improve the efficacy and outcomes of cancer chemotherapy. CSE1L expression in cancer CSE1L is highly expressed in various cancer types, and its expression level is positively correlated with high tumor stage, high tumor grade, and worse outcomes of cancer patients. The CSE1L gene is located on chromosome 20q13, a region frequently harbors amplifications that correlate with cancer aggression [33–35]. The copy number of the CSE1L gene is increased in breast, colon, and bladder cancer cell lines [36]. An array-based comparative genomic Vorinostat hybridization study showed high-frequency amplifications of the CSE1L gene in nasopharyngeal carcinomas [37] and in medulloblastomas [38]. The results of array-based comparative genomic hybridization showed that 57.1% of the glioblastoma multiforme cases had high-frequency amplification of the CSE1L gene [39]. Idbaih et al. investigated a series of 16 low-grade gliomas and their subsequent progression to higher-grade

malignancies using a one-megabase bacterial artificial chromosome (BAC)-based array comparative genomic hybridization technique, and reported PRKACG that the CSE1L gene was associated with the progression of gliomas [40]. The results of another study using microarray-based detection showed that CSE1L was highly expressed in nasopharyngeal carcinomas [41]. Combined cytogenetic, array-based comparative genomic hybridization studies and expression analyses also showed that CSE1L was significantly overexpressed in advanced prostate cancer xenografts [42]. The results of a pathological study showed that expression of CSE1L was not detected in normal hepatocytes, while strong CSE1L expression was detected in hepatocellular carcinoma [10].

Leriche et al [19] have described the protection of certain bact

Leriche et al. [19] have described the protection of certain bacterial strains by other strains within a mixed biofilm system. We therefore investigated the potential for a “”non biofilm-forming”" isolate (isolate 80) to be incorporated into the biofilm produced by isolate 17, a strong biofilm producer and showed that not only can an established biofilm of P. aeruginosa assist in the attachment and colonisation of another isolate, but also that the two P. aeruginosa isolates became integrated in a mixed biofilm as shown in cross section CSLM images (Fig. 5). In the mixed

biofilm scenario in vitro, the CF P. aeruginosa biofilm could consist of many different isolates, some of which are unable to form biofilms themselves yet can colonise an already established biofilm. Adaptability this website is the key to successful colonisation of an environmental niche and in the field of infectious disease, it is widely accepted that NVP-BGJ398 price a pathogen will normally have more than one way of exerting a pathogenic effect. Many pathogens, therefore, have multiple adhesion mechanisms allowing attachment to, for example, epithelial cells [46]. We contend that the LY2874455 cell line physiological mechanisms involved in biofilm formation should be considered in a similar manner,

in that a deficiency in one phenotypic aspect of biofilm formation may be compensated for by other genetic and phenotypic factors. Conclusions Motility makes a positive contribution to biofilm formation in CF isolates of P. aeruginosa, but is not an absolute requirement. It is clear that CF isolates with differing motility phenotypes can act synergistically to form a mixed biofilm. This could give an advantage to bacterial communities as they would possess a greater repertoire of genetic ability, thus allowing them to adapt to different challenges e.g. antibiotic chemotherapy,

host inflammatory responses, etc. Acknowledgements ED was in receipt of a Vice Chancellor’s Research Scholarship from the University of Ulster. ED also gratefully acknowledges receipt of a Society for General Microbiology “”President’s Fund”" award for travel to the CBE, MT, USA. Thanks are due to Dr Graham Hogg (Belfast City Hospital) for providing the P. aeruginosa strains used in this study Aurora Kinase to and Dr Steven Lowry of the University of Ulster for his assistance with SEM. We thank Prof. Phil Stewart for the hospitality in his laboratory at The Centre for Biofilm Engineering, MT and Ms Betsy Pitts for providing training and assistance with CSLM studies. References 1. Lawrence JR, Horber DR, Hoyle BD, Costerton JW, Caldwell DE: Optical sectioning of microbial biofilms. J Bacteriol 1991, 173:6558–6567.PubMed 2. Nickel JC, Costerton JW: Bacterial localisation in antibiotic-refractory chronic bacterial prostatitis. Prostate 1993, 23:107–114.

J Clin Microbiol 2001, 39:4227–4232 CrossRefPubMed 2 Woo PC, Kuh

J Clin Microbiol 2001, 39:4227–4232.CrossRefPubMed 2. Woo PC, Kuhnert P, Burnens AP, Teng JL, Lau SK, Que TL, Yau HH, Yuen KY:Laribacter hongkongensis : a potential cause of infectious Selleckchem Capmatinib diarrhea. Diagn Microbiol Infect Dis 2003, 47:551–556.CrossRefPubMed 3. Lau SK, Woo PC, Hui WT, Li MW, Teng JL, Que TL, Luk WK, Lai RW, Yung RW, Yuen KY: Use of cefoperazone MacConkey

agar for selective isolation of Laribacter hongkongensis. J Clin Microbiol 2003, 41:4839–4841.CrossRefPubMed 4. Woo PC, Lau SK, Teng JL, Que TL, Yung RW, Luk WK, Lai RW, Hui WT, Wong SS, Yau HH, Yuen KY: Association of Laribacter hongkongensis in community-acquired human gastroenteritis with travel and with eating fish: a multicentre case-control study. Lancet 2004, 363:1941–1947.CrossRefPubMed 5. Ni XP, Ren SH, Sun JR, Xiang HQ, Gao Y, Kong QX, Cha J, Pan JC, Yu H, Li HM:Laribacter hongkongensis isolated from a community-acquired gastroenteritis in Hangzhou City. J Clin Microbiol 2007, 45:255–256.CrossRefPubMed 6. Marcos LA, DuPont HL: Advances in defining etiology and new therapeutic approaches

in acute diarrhea. J Infect 2007, 55:385–393.CrossRefPubMed 7. Farmer JJ, Gangarosa RE 3rd, Gangarosa EJ: Does Laribacter hongkongensis cause diarrhoea, or does diarrhoea “”cause”" L hongkongensis? Lancet 2004, 363:1923–1924.CrossRefPubMed 8. Lau SK, Woo PC, Fan RY, Lee RC, Teng JL, Yuen KY: Seasonal and tissue distribution of Laribacter hongkongensis , a novel bacterium associated with gastroenteritis, in retail freshwater fish in Hong Kong. Int J Food Microbiol 2007, 113:62–66.CrossRefPubMed Edoxaban 9. Teng JL, Woo C646 in vivo PC, Ma SS, Sit TH, Ng LT, Hui WT, Lau SK, Yuen KY: Ecoepidemiology of Laribacter hongkongensis , a novel bacterium associated with gastroenteritis. J Clin Microbiol 2005, 43:919–922.CrossRefPubMed 10. Lau SK, Woo PC, Fan RY, Ma SS, Hui WT, Au SY, Chan LL, Chan JY, Lau AT, Leung KY, Pun TC,

She HH, Wong CY, Wong LL, Yeun KY: Isolation of Laribacter hongkongensis , a novel bacterium associated with gastroenteritis, from drinking water reservoirs in Hong Kong. J Appl Microbiol 2007, 103:507–515.CrossRefPubMed 11. Hall TA: BioEdit: a user-friendly biological see more sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 1999, 41:95–98. 12. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673–4680.CrossRefPubMed 13. Jolley KA, Chan MS, Maiden MCJ: Sequence type analysis and recombinational tests (START). Bioinformatics 2001, 17:1230–1231.CrossRefPubMed 14. Didelot X, Falush D: Inference of bacterial microevolution using multilocus sequence data. Genetics 2007, 175:1251–1266.CrossRefPubMed 15. Tamura K, Dudley J, Nei M, Kumar S: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.

Ratios of phospho-FAK to total FAK and total FAK to control bands

Ratios of phospho-FAK to total FAK and total FAK to control bands were also normalized to dormant cells. b GRAF membrane localization in dormant cells and the ATM inhibitor corresponding RhoA departure form its membrane localization was demonstrated on immunofluorescence-stained cells on fibronectin-coated cover slips (red) and photography at 630 x magnification. Growing cells exhibited membrane localization of RhoA (arrows) which disappeared in dormant cells,

while GRAF membrane localization appeared in dormant cells (arrows). Immunostaining with antibody to p190 Rho GAP was used as a negative control, demonstrating no evident staining in either growing or dormant cells. Nuclear DAPI staining is shown in blue. c Membrane fractionation of growing and dormant cells with and without added blocking antibodies to integrin α5β1 and integrin α2β1 2 μg/ml and western blotting of isolates with antibody to GRAF and BAX, used as a cytoplasm-localizing control. Bands were quantitated using a densitometer and ratios of membrane- to cytoplasm-localizing GRAF and BAX

were calculated To determine a possible mechanism for the inactivation of RhoA in dormant cells, we analyzed the FAK immunoprecipitates for GTPase Regulator Associated with the Focal Adhesion Kinase pp125(FAK) (GRAF), a protein with demonstrated RhoA GAP activity shown to co-localize with activated FAK in focal BMN 673 in vitro complexes. Figure 6a suggests SN-38 in vitro that GRAF becomes associated with FAK in dormant cells, an effect exclusively dependent on integrin α5β1. To confirm this result, we analyzed the cells by immunfluorescence. Figure

6b demonstrates that GRAF became membrane localized in the dormant cells in a reciprocal relationship to the loss of RhoA membrane localization. As a control, Fig 6b demonstrates that the RhoA GAP p190 was not affected in dormant cells. To further confirm the activation by membrane localization of GRAF in dormancy, we carried out membrane fractionation experiments. Figure 6c demonstrates that GRAF was primarily cytoplasm localized in growing cells with a membrane to cytoplasm (m/c) ratio of 0.25. GPX6 In dormant cells, GRAF membrane localization increased to an m/c ratio of 0.61. This effect once again was dependent on integrin α5β1, as blocking antibody to this integrin decreased the ratio to 0.15. With blocking antibody to integrin α2β1 used as a control, the GRAF m/c ratio was 0.80. These data support the hypothesis that the RhoA GAP GRAF becomes activated and membrane localized in dormant cells causing an inactivation of RhoA and that this effect depends on binding of integrin α5β1. Activation of PI3K is Independent of Integrins α5β1 Binding in Dormant Cells We have previously demonstrated that the PI3K pathway is activated in these dormant cells [3]. This activation is sustained for the 5 days assayed and its inhibition blocked survival of the dormant clones.