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H. Nmr,

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, HPLC: t R = 20.9 min, purity >93%. MS(ESI+): m/z (%): 279.13 (100

, 829 cm ?1 . 1 H NMR (300 MHz, 65% yield) as a white solid. IR 2931, 1234.

. Hz,

C. Nmr,

, mL) was added to the previous solution, and the resulting reaction mixture was stirred for 12 h at rt and 1 h under reflux (for a complete conversion). Thereafter, the mixture was cooled to rt and concentrated. The residue was filtered over a silica gel pad (cyclohexane/EtOAc 1:1 then EtOAc) to afford 8 (73 mg, 0.15 mmol, 60%) as a white solid, The resulting mixture was stirred at 0°C for 15 min and at rt for further 15 min. A solution of the benzyl bromide 2 (97 mg, 0.28 mmol, 1.1 equiv) in dry DME, vol.7, p.1228, 1599.

D. +27°, , vol.3

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D. +27°, HRMS (ESI+): Calc. for C 27 H

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, Brain Function PLOS ONE, 20142-12-01.

, This project was approved by the ''Comité d'Ethique NOrmandie en Matière d'EXpérimentation Animale'' CENOMEXA under the National Committee on Animal Experimentation, and received the following number N/ of everolimus formulated at 2% (w/v), provided by Novartis (Rueil-Malmaison, France), was dissolved in 0.9% NaCl and administered daily by oral gavage (5 mL/kg) via a feeding needle (Fine Science Tools, Heidelberg, anesthetized using isofluorane, decapitated, and their brains rapidly removed 24 hours after the last day of treatment, Ethical Committee as well as the guidelines of European Parliament directive 2010/63/EU and the Council for the Protection of Animals Used for Scientific Purposes

, ) and microscopically examined. The product of the histochemical reaction (oxidized DAB) was visible with optical microscopy. In each area of interest, its staining intensity was measured by densitometric analysis by means of a computer-assisted image analysis workstation, 300 mg DAB-4-HCl, and 108 mg catalase, p.37

P. One and . Doi, , 20144-12-01.

, Sections were then washed (PBS, 365 minutes), incubated for 1 hour in blocking solution containing 1:50 normal donkey serum, 1% bovine serum albumin, and 0.3% Triton X-100 (VWR International, PBS, and incubated overnight at 4?C with sheep anti-BrdU immunoglobulin G

. Abcam, 1:400 in PBS. Rinsed sections were cover slipped with mowiol. Sections were examined on a Nikon Eclipse E600 microscope (Nikon Instrument, Champigny-sur-Marne, France) interfaced with the Mercator software (ExploraNova, La Rochelle, France). group. For investigation of hippocampal vascular niche density, brain sections were fixed, washed, and incubated as described above, 1:400 in blocking solution. Subsequently, sections were washed (PBS, 465 minutes) and incubated (2 hours, room temperature) with Alexa 488-conjugated donkey antisheep IgG (Invitrogen, vol.1, 2014.

. Neurobasal-;-ml, . Gibco, and . Saint-aubin, 1 mL B27 and 1 mL N2 growth supplement (Gibco), 20 mL epidermal growth factor, France) supplemented with 5 mM HEPES buffer

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P. One and . Doi, , 2014.

P. One and . Doi, , 2014.

P. One and . Doi, , 2014.

, Percentage of optical densities of cytochrome oxidase activity staining after everolimus treatment in the telencephalon, the diencephalon, the mesencephalon, the metencephalon, the myelecephalon and the cerebellum. Data are means +SEM. (Student t test, *p,.05). (a, area; ant, anterior; Diag, diagonal; horiz, horizontal; genic, geniculate; mesenc, mesencephalic; n, nucleus; ret, reticular; vert, vertical), Brain Function PLOS ONE, vol.13, p.27, 2014.

, Figure 2. Phospho-P70S6K labeling in brain regions of everolimus and vehicle mice. (a) CO activity in selective vehicle-and everolimus-treated mice brain areas. (b) Representative images of Phospho-P70S6K (p-P70S6K) immunolabeling in cortex, entopeduncular nucleus, thalamus and hypothalamus of vehicle or everolimus-treated mice. (c) Mean percent of P-P70S6K-labeled surface (normalized to vehicle) (+SEM) in the different brain areas. Arc: arcuate nucleus; Ect: ectorhinal cortex; EP: entopeduncular nucleus; PRh: perirhinal cortex; PSTh: parasubthalamic nucleus; Re: reuniens thalamic nucleus; Rh: rhomboid thalamic nucleus; Submed: submedial thalamic nucleus; VM: ventromedial thalamic nuleus, Brain Function PLOS ONE, vol.14, p.27, 2014.

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, The glioma cell lines SW1088 and U87 were from American Type Culture Collection (ATCC). SW1783 cell line was given by, ng/mL) and 0.4% Methyl-cellulose

, Plasmid constructs, siRNA and transfection The human UT receptor cDNA into pcDNA3.1 (cDNA Ressource Center, MI, USA) was subcloned into pCMV-c-myc (BD Biosciences), peGFP-N2, pmCherry-N2 and peYFP-N1 provided by Dr O. Wurtz (Inserm U982

, Roche Diagnostics) to introduce three Ala instead of Glu 147 , Arg 148

, G ?i fused to RLuc or to eYFP, G? fused to eYFP and G ?13 fused to eYFP were provided by

L. Dr and . Prézeau, G ?13 -RLucII was prepared as described. 69 Control or G ?13 siRNA (5'GUUCAUGGUAUCGUGCAUGdTdT3'; 70 ) were from Dharmacon

, Plasmid DNAs transient transfections were performed using Lipofectamine?, 2000.

, Urantide TM was from Peptides International, Palosuran was provided by Actelion Pharmaceuticals (Allschwill, Suisse). PTX, Y27632 or, p.294002

(. Kit and . Dako, Danemark) according to the manufacturer's instructions. Primary rabbit antihuman UII (1/50, Sigma Aldrich), rabbit anti-UT (1/100, Santa Cruz Biotechnology, France) and mouse anti-human CD34 (1/50, Leica Biosytems, France) were used. Nuclei were counterstained with hematoxylin solution

, Flow Cytometry Glioma cells were pelleted and incubated with non-immune rabbit IgGs (10 µg/mL, vol.30

. Bsa, After permeabilization with saponin (0.1%, 30 min), cells were incubated with antibodies directed against UT (2 µg/mL, Santa Cruz) or UII (10 µg/mL, Phoenix Pharmaceuticals) at RT for 30 min, /200), cells were analyzed on a FACScalibur flow cytometer

, Immunocytochemistry Glioma cells were fixed in PFA (4%, 10 min)

, Cells were incubated overnight with primary antibodies (Anti-UT, 0.5 µg/mL

. Anti-?3-integrin,

, Abcam) at 4°C followed by incubation for 2h at RT with secondary fluorescent antibodies (Alexa 488 or 594 conjugated antibodies, 1/200, Invitrogen). Finally, cells were counterstained with DAPI (Invitrogen, 1 µg/mL, 10 min), and imaged by confocal microscopy (Leica TCS SP5 confocal laser scanning microscope). For F-actin staining

, 1X protease and/or phosphatase inhibitor cocktails), mixed with Laemmli buffer and loaded onto 10% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane. Membranes were blocked with 2% nonfat milk or 4% BSA, incubated with the primary UT (Abcam 78449, 0.5µg/ml) or actin antibody (Abcam 1801, 1 mg/ml), overnight at 4°C, then with HRP-conjugated secondary antibodies, Western blotting Cell lysates were prepared in ice-cold Lysis Buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10% NP40

G. Ut and . ?1, The G ?i/seeded on fibronectin-coated transwell filters

, Migrating cells were fixed, stained with eosin/hematoxilin and filters were mounted on glass slides with Mowiol and randomly microphotographed with a Nikon microscope (Champignysur-Marne, France, 10 fields/filter). 30 min (Molecular probes), resuspended in serum-free medium and treated (37°C, 30 min). Cells were plated on fibronectin-coated 96-well plates and washed twice with PBS after 15 (HEK) or 30 min (SW1088), and adherent cells were measured with a fluorescence plate reader, vol.600

, Glioma Cells were plated into fibronectin-coated glass bottom dish (MatTek corporation, Ashland, USA) in medium supplemented with FBS and co-transfected with cDNAs of UT mCherry and G ?i eYFP or G ?13 eYFP

, Images were collected every 30s on a videomicroscope (Leica, DM IRE2), and UII was added

, Ca 2+ ] c , cytosolic calcium concentration

A. -. Aobs, Optical Beam Splitter; ATCC, American Type Culture Collection; bFGF, basic fibroblast growth factor; BRET, bioluminescence resonance energy transfer

, Central Brain Tumor Registry of the United States; CCD, charge-coupled device, CBTRUS

, CNS, central nervous system

, CXCR4, CXC motif receptor 4

, DAPI, 4',6'-diamidino-2-phenylindole

, EGF, epidermal growth factor

, FA, focal adhesion; FBS, fetal bovine serum; FRET, fluorescence resonance energy transfer

G. Gbm and . Multiforme,

G. Gpcr and . Protein,

, HRP

, HTRF, homogeneous time-resolved fluorescence; IP-1, inositol phosphate; NDS, normal donkey serum; NEAA, non essential amino acids; NPY, neuropeptide Y; PFA, paraformaldehyde; PI, propidium iodide, vol.3, p.3

. Pip and . Plc,

P. Ptx and . Toxin,

. Rluc,

. Rb,

R. Rnai and . Interference,

, rho kinase; s.c., subcutaneously; shRNA, small hairpin RNA; TMRM, tetramethylrhodamine

U. Uii and . Ii,

, URP, urotensin II-related peptide; UT, urotensin II receptor

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. Ki67>10% and . Left, Pink labelings indicate CD34-positive features. Left, middle and right represent adjacent sections. Scale Bars, 50 µm. (b) Immunoreactivity of UT in a fresh GBM tumoral explant, showing strong green labeling associated to a number of tumoral cells (a, z = 0 µm), vascular components (b, z = 47 µm) and invading cells emerging from the explants (c, z = 114 µm). (c) Flow cytometry analysis of UT (upper) and UII (lower) expression levels in human astrocytoma grade III (SW1783 and SW1088), eosin/hematoxylin staining showing characteristic necrotic area (*), p.15

. Sw1783, of UII (10 -9 M, 24h) added in the bottom well. (a, b) and Whitney test, ns

*. and P. ,

P. **,

. ***, SW1088 were exposed to the antagonists palosuran and urantide, respectively (10 -5 M each) added both in the upper and bottom chambers in the absence or the presence of UII, HEK-UT ERY(AAA) ) were plated in the top chamber and UII, pp.10-19

, (e) BRET between G ?i and G ??i or UT and G ?13 in HEK293 co-expressing ?i-Rluc and ??-eYFP or UT-RlucII and ?13-eGFP2 in the presence of increasing concentrations of UII

W. Mann and . Test,

*. and P. , 001 versus control (in the absence of UII). (f) and Whitney test, *

P. **, 01. (g) in quadruplicates. Mann and Whitney test, ns, no significant

P. **, 05. (h) SW1088 were exposed in the Boyden chamber to the ROCK inhibitor Y27632 and the PI3K inhibitor LY294002, respectively (10 -5 M each) pre-incubated (6h) and and Whitney test, ns

P. **, 01. (i) Regulation of Rho small GTPase by UII in SW1088. SW1088 were treated in the absence (control), the presence of UII (10 -8 M; 10 min) or FBS. Western blot assay showed the expression of total and activated Rho

, Immunofluorescence of SW1088-UT eGFP (green) and actin labeled with a phalloidinrhodamine staining (red). SW1088 were non-transfected (SW1088) or transfected with cDNAs encoding UT eGFP (SW1088-UT eGFP ) pre-treated 6h with LY294002, Y27632 or PTX and incubated in the absence (control) or the presence of UII (10 recombinant SW1088-UT eGFP, Figure 3. UII effect on actin polymerization and lamellipodia formation in glioma cells. (a)

*. and P. ,

P. **,

. ***, Living cells were imaged in real time by using time-lapse video P<0.01. (d) Adhesion assay of SW1088 preincubated with palosuran or urantide, p.30

, min, each) in the absence or the presence of UII

, Multiple Comparison Test, **, P<0.01. (e) Adhesion assay of SW1088 preincubated (6h) with

, Bar graph represents mean ± SEM obtained from 2 independent experiments in quintuplets. Mann and Whitney test, ns, PTX (0.2 µg/µl), Y27632 (10 -5 M) or LY294002 (10 -5 M) and exposed to UII (10 -8 M)

P. **,

. ***, Adhesion assay on fibronectin of HEK293 transfected with the cDNA encoding c-myc (HEK), UT c-myc (HEK-UT) or UT ERY(AAA) c-myc (HEK-UT ERY(AAA) ) in the absence (control) or the presence of UII (10 -and Whitney test, ns

P. **, 01. (g) Dynamic distribution of G ?i eYFP proteins and UT mcherry in SW1088 before

, min) and after treatment with UII (T=1, 15 and 30 min)

, In SW1088, the G ?i and UT co-localization (in yellow) was revealed in plasma membrane (white arrow head), glioma. SW1088 were non-transfected (SW1088) or transfected with cDNAs encoding UT eGFP (SW1088-UT eGFP ) and incubated in the absence (control) or the presence of inhibitors (LY294002, Y27632 or PTX) and treated with UII, individual frames were shown before and during UII application

, Scale bars indicates 25 µm. (c and d) Quantification of focal adhesion points per cell (c) and distribution of focal adhesion area (d) in SW1088 or recombinant SW1088-UT eGFP exposed to UII (10 -8 M, 1h) in the absence or the presence of inhibitors (LY294002, Y27632 or PTX). Mann and Whitney test, ns

*. , , p.0