This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1. Préparer des solutions siARN <ol><li> Remettre en suspension 5 nmol de brouillés (Ctrl) et 5 nmol de pastilles séchées BRCA2 siRNA dans 100 ul d&#39;eau exempte de RNase (concentration finale de siRNA: 50 um). Ceci est le stock siRNA et doit être stocké à -20 ° C en 10 aliquotes. </li><li> Prendre une aliquote de 10 pi de stock siRNA et ajouter 40 ul d&#39;eau sans RNase pour obtenir la solution de travail 10 uM siRNA. Cette dilution doit être conservé à -20 ° C jusqu&#39;à utilisation. NOTE: La solution de travail siRNA ne doivent pas subir de congélation / décongélation plus de 3 fois. </li></ol> 2. Ensemencement des lignées cellulaires épithéliales humaines <ol><li> Retirer le moyen de lignées cellulaires epitheliales humaines en croissance sous forme de monocouches de cellules dans des plaques de culture tissulaire dans un incubateur humidifié de croissance à 37 ° C, 5% CO 2. </li><li> Laver les cellules dans la plaque une fois avec du PBS (RT 10 ml de PBS pour une plaque de 100 mm). </li><li> Pour détacher les cellules de la plaque,ajouter 2 ml de trypsine à 0,25% / solution 0,53 mM d&#39;EDTA et incuber 3-5 min en fonction du type de cellule. </li><li> Avant de collecter les cellules détachées, neutraliser la trypsine par addition de 2 ml de milieu de croissance complet contenant 10% de sérum bovin foetal. </li><li> Transférer les cellules dans un tube de 15 ml. </li><li> Centrifuger les cellules à 320 à 330 g pendant 3 min à 4 ° C dans un rotor à godets oscillants. </li><li> Remettre en suspension les culots cellulaires dans 5 ml de milieu sans antibiotiques. </li><li> Compter les cellules au microscope dans une chambre Burker ou en utilisant un système de comptage automatique selon les instructions du fabricant. </li><li> Ensemencer les cellules dans des plaques de 6 puits à environ 80% de confluence après 24 h (0,5-1 x10 6 cellules dans 2 ml de milieu de chaque puits). Le nombre de cellules optimal à être ensemencée peut varier en fonction des lignées cellulaires spécifiques et le taux de croissance. NOTE: Ne pas ajouter des antibiotiques dans le milieu à ce point parce que les antibiotiques vont interférer négativement avec l&#39;efficacité de transfection. Il est très important que les cellules sont à passages en culture faible (&lt;10) pour atteindre la haute efficacité de transfection de siRNA BRCA2. </li></ol> 3. transfecter des cellules avec un siRNA <ol><li> Vingt-quatre heures après l&#39;étalement des cellules, procéder à la première ronde de la transfection. <ol><li> Diluer 6 pi de 10 réactif de transfection de siRNA spécifique à base de lipides dans 130 ul de milieu de sérum réduit. </li><li> Diluer 3 pi de 10 pM de siRNA (30 pmoles) dans 130 ul de milieu de sérum réduit. </li><li> Moissonneuse le siRNA dilué avec le réactif de transfection dilué et incuber pendant 5-10 min à température ambiante. </li><li> Pendant l&#39;incubation, éliminer le milieu à partir des cellules et ajouter 2 ml de milieu frais préchauffé à 37 ° C (sans antibiotiques). </li><li> Ajouter 250 ul de complexes réactif de transfection-ARNsi aux cellules (cette quantité est d&#39;un seul puits d&#39;une plaque à 6 puits). </li><li> Incuber les cellules dans un incubateur humidifié à 37 ° C76; C avec 5% de CO 2. </li></ol></li><li> Après 24 h, passer à la deuxième ronde de la transfection en répétant les étapes 3.1.1-3.1.5 avec la modification suivante pour l&#39;étape 3.1.2: diluer 5 pi de 10 uM siRNA (50 pmol) dans 130 pi de milieu sérique réduite. <ol><li> Incuber les cellules pendant 1-2 jours à 37 ° C avant analyse. REMARQUE: Deux séries de transfections et utilisation d&#39;un rapport de réactif haute siRNA / de transfection au second tour sont essentiels pour obtenir un rendement élevé de BRCA2 silencieux. </li></ol></li></ol> 4. Lyse des cellules pour l&#39;analyse d&#39;immunotransfert <ol><li> Préparer du tampon de lyse douce avec du PBS (pH 7,4) contenant 1% de la non-ioniques, non dénaturant détergent octylphénoxypolyéthoxyéthanol, le pyrophosphate de sodium 5 mM, orthovanadate de sodium 1 mM, fluorure de sodium 10 mM, fluorure de phénylméthylsulfonyle 2 mM, 10 ug / ml de leupeptine, 10 ug / ml d&#39;aprotinine. Gardez-le sur la glace. </li><li> Enlever le milieu à partir des cellules et wles cendres une fois avec du PBS froid (2 ml / puits) dans la plaque. Pour éviter tout report PBS, retirer complètement de la plaque. </li><li> Ajouter 200 ul de tampon de lyse dans chaque puits. REMARQUE: si les cellules doivent être utilisés aussi pour d&#39;autres dosages, trypsiniser les cellules comme décrit dans l&#39;étape 2/1 à 2/6 et remettre en suspension les cellules dans du tampon / milieu approprié selon l&#39;application à utiliser. </li><li> Faire tourner doucement la plaque pour distribuer uniformément le tampon de lyse et incuber pendant 15 min à 4 ° C sur un agitateur. </li><li> Recueillir le lysat cellulaire dans un tube de microcentrifugeuse sur de la glace en utilisant un grattoir à cellules. </li><li> Centrifuger à 17 900 g pendant 25 min à 4 ° C et recueillir le surnageant dans un nouveau tube. </li><li> Mesurer la concentration de protéine en utilisant un dosage de protéine disponible dans le commerce selon les instructions du fabricant. </li></ol> Analyse d&#39;immunotransfert 5. BRCA2 <ol><li> Préparer le tampon de migration en diluant 50 ml de 20x Tris-Acétate SDS b couriruffer (mM Tricine 50, 50 mM Tris base, 0,1% de SDS, pH 8,24) avec 950 ml d&#39;eau distillée. </li><li> Préparer l&#39;échantillon comme suit: ajouter 15 pg de lysat de cellules totales, 5 ul de tampon d&#39;échantillon 4x contenant dodécylsulfate de lithium à un pH de 8,4, 2 pl de 10x échantillon agent réducteur (0,5 M DTT), et un tampon de lyse jusqu&#39;à un volume final de 20 ul. NOTE: Ce protocole permet la détection de BRCA2 dans des lignées cellulaires normales aussi lors de l&#39;utilisation d&#39;une plus faible quantité de protéines totales (en bas à 5 ug). </li><li> Dénaturer les échantillons à 55 ° C pendant 10 min. NOTE: Il est crucial d&#39;utiliser 55 ° C. Depuis protéine BRCA2 est thermosensible 11, la hausse des températures de dénaturation se traduisent par la perte constante de pleine longueur intacte (390 kDa) des protéines BRCA2. </li><li> Mettre en place la chambre électrophorétique selon les instructions du fabricant. </li><li> Charger les échantillons et 10 pi de haut poids moléculaire pré-coloré protéine standard sur un gel precasted (Tris-Acétate 3-8%) et de fonctionner à 120 V pour abà 2 h 12. Arrêter la course électrophorétique avant la kDa marqueur bleu 55 laisse le gel precasted. </li><li> Préparer le tampon de transfert avec 50 ml de tampon de transfert 20 fois, 100 ml de methanol à 100% et 850 ml d&#39;eau. </li><li> Activer membrane de PVDF (0,45 um de taille de pore) par trempage dans 100% de methanol pendant 5 minutes, rincer à l&#39;eau distillée et équilibrer dans du tampon de transfert au moins pendant 5 minutes. Faire tremper les éponges de l&#39;appareil de transfert dans un tampon de transfert à 4 ° C jusqu&#39;à utilisation. </li><li> Lorsque la course électrophorétique est terminée, assembler le sandwich dans la pile de transfert dans l&#39;ordre suivant (bottom-up): trois éponges saturées dans un tampon de transfert, un 3MM feuille de papier de qualité saturée dans un tampon de transfert, le gel, la membrane de PVDF activé, un 3MM Note feuille de papier saturée dans un tampon de transfert, trois éponges saturée dans un tampon de transfert 13,14. Placez la pile dans les protéines de l&#39;appareil et de transfert à 350 mA pendant 4 heures à 4 ° C ou à 180 mA pendant une nuit à 4 ° C. NOTE: Etre BRCA2 une très grosse protéine, il est crucial d&#39;étendre le temps de transfert de 1 h (comme suggéré par le fabricant et la plupart des protocoles de transfert) à 4 h ou toute la nuit pour assurer le transfert complet des protéines de haut poids moléculaire. En outre, en raison de la longue période de transfert, il est essentiel d&#39;effectuer le buvard dans une chambre froide à 4 ° C pour minimiser la surchauffe et de faciliter le démontage en sandwich. Systèmes de transfert semi-sèches ne sont pas bonnes pour transférer de grandes protéines. </li><li> Après le transfert, laver la membrane avec le SCT, bloquer pendant 1 heure à température ambiante dans du TBS-Tween 0,25% (TBS-T) contenant du lait en poudre de 5% sans matières grasses et de la sonde pendant la nuit à 4 ° C avec un anticorps polyclonal de lapin 30 pi anti-BRCA2 ( 200 ug / ml), dilué dans 9 ml de TBS-T + 5% de lait écrémé sec [1: 300 (v / v)] dilution. </li><li> Laver la membrane trois fois (10 minutes à chaque fois) avec du TBS-T, puis incuber le filtre pendant 1 heure avec 1 pi d&#39;anticorps secondaire conjugué à la peroxydase de raifort anti-lapin dilué dans 10 ml de TBS-T + 5% de lait écrémé sec. Par la suite, laver la membrane trois fois (10 minutes chacun) avec TBS-T. </li><li> Effectuer immunodétection par ECL pour révéler la protéine BRCA2 utilisant la chimioluminescence amplifiée (ECL) Western Blot réactif de détection, en suivant les instructions du fabricant. Visualiser les bandes immunochemiluminescent sur haute résolution ECL films. </li><li> Effectuer immunodétection avec un anticorps pour un gène de ménage, comme β-tubuline (55 kDa), sur le même filtre que le contrôle de chargement en utilisant un anticorps monoclonal à β-tubuline dilué à 1: 1000 (10 ul d&#39;anticorps anti-tubuline en 10 ml TBS-T + 5% de lait sec non gras) pendant 1 heure à température ambiante. Laver et incuber l&#39;anticorps secondaire tel que décrit pour BRCA2 (5.10 à 5.11), à l&#39;exception de l&#39;utilisation de la peroxydase de raifort anti-souris conjuguée à la place de l&#39;anticorps secondaire anti-lapin. </li><li> Acquérir une image numérique de films impressionnés par bandes de immunochemiluminescent et analyser l&#39;intensité de la bande par un logiciel d&#39;analyse d&#39;image spécialisé (par exemple,., ImageJ). </li></ol>

<ol>
	<li>Thorslund, T., West, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA2:+a+universal+recombinase+regulator.">BRCA2: a universal recombinase regulator.</a> Oncogene. 26, 7720-7730 (2007).</li><li>Yoshida, K., Miki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+BRCA1+and+BRCA2+as+regulators+of+DNA+repair,+transcription,+and+cell+cycle+in+response+to+DNA+damage.">Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage.</a> Cancer Sci. 95, 866-871 (2004).</li><li>Martin, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+mutations+in+BRCA1+and+BRCA2+in+breast-ovarian+families+from+a+breast+cancer+risk+evaluation+clinic.">Germline mutations in BRCA1 and BRCA2 in breast-ovarian families from a breast cancer risk evaluation clinic.</a> J Clin Oncol. 19, 2247-2253 (2001).</li><li>Castro, E., Eeles, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+BRCA1+and+BRCA2+in+prostate+cancer.">The role of BRCA1 and BRCA2 in prostate cancer.</a> Asian J Androl. 14, 409-414 (2012).</li><li>Vencken, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensitivity+and+outcome+of+BRCA1-+and+BRCA2-associated+ovarian+cancer+patients+after+first-line+chemotherapy+compared+with+sporadic+ovarian+cancer+patients.">Chemosensitivity and outcome of BRCA1- and BRCA2-associated ovarian cancer patients after first-line chemotherapy compared with sporadic ovarian cancer patients.</a> Ann Oncol. 22, 1346-1352 (2011).</li><li>Farmer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+DNA+repair+defect+in+BRCA+mutant+cells+as+a+therapeutic+strategy.">Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.</a> Nature. 434, 917-921 (2005).</li><li>Arbini, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+depletion+sensitizes+cancer+cells+to+PARP+inhibitors+by+translational+and+post-translational+repression+of+BRCA2.">Mitochondrial DNA depletion sensitizes cancer cells to PARP inhibitors by translational and post-translational repression of BRCA2.</a> Oncogenesis. 2, e82 (2013).</li><li>Yang, F., Guo, X., Yang, G., Rosen, D. G., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AURKA+and+BRCA2+expression+highly+correlate+with+prognosis+of+endometrioid+ovarian+carcinoma.">AURKA and BRCA2 expression highly correlate with prognosis of endometrioid ovarian carcinoma.</a> Mod Pathol. 24, 836-845 (2011).</li><li>Guaragnella, N., Marra, E., Galli, A., Moro, L., Giannattasio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+BRCA2+decreases+anoikis+and+its+heterologous+expression+sensitizes+yeast+cells+to+acetic+acid-induced+programmed+cell+death.">Silencing of BRCA2 decreases anoikis and its heterologous expression sensitizes yeast cells to acetic acid-induced programmed cell death.</a> Apoptosis. 19, 1330-1341 (2014).</li><li>Mahato, R. I., Rolland, A., Tomlinson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-based+gene+delivery+systems:+pharmaceutical+perspectives.">Cationic lipid-based gene delivery systems: pharmaceutical perspectives.</a> Pharm Res. 14, 853-859 (1997).</li><li>Su, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+BRCA2:+temperature+sensitivity+of+detection+and+cell-cycle+regulated+expression.">Characterization of BRCA2: temperature sensitivity of detection and cell-cycle regulated expression.</a> Oncogene. 17, 2377-2381 (1998).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76, 4350-4354 (1979).</li><li>Burnette, W. N. '. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Western+blotting':+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Anal Biochem. 112, 195-203 (1981).</li><li>Hunter, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+targeting+of+hypoxia+and+homologous+recombination+repair+dysfunction+in+triple-negative+breast+cancer.">Dual targeting of hypoxia and homologous recombination repair dysfunction in triple-negative breast cancer.</a> Mol Cancer Ther. , (2014).</li><li>Drew, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+poly(ADP-ribose)+polymerase+inhibitor+AG014699+in+human+cancers+with+mutated+or+methylated+BRCA1+or+BRCA2.">Therapeutic potential of poly(ADP-ribose) polymerase inhibitor AG014699 in human cancers with mutated or methylated BRCA1 or BRCA2.</a> J Natl Cancer Inst. 103, 334-346 (2011).</li><li>Bryant, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+killing+of+BRCA2-deficient+tumours+with+inhibitors+of+poly(ADP-ribose)+polymerase.">Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.</a> Nature. 434, 913-917 (2005).</li><li>Ihnen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+the+poly(ADP-ribose)+polymerase+inhibitor+rucaparib+for+the+treatment+of+sporadic+human+ovarian+cancer.">Therapeutic potential of the poly(ADP-ribose) polymerase inhibitor rucaparib for the treatment of sporadic human ovarian cancer.</a> Mol Cancer Ther. 12, 1002-1015 (2013).</li><li>Jeong, J. H., Jo, A., Park, P., Lee, H., Lee, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brca2+Deficiency+Leads+to+T+Cell+Loss+and+Immune+Dysfunction.">Brca2 Deficiency Leads to T Cell Loss and Immune Dysfunction.</a> Mol Cells. , (2015).</li></ol>

The authors have nothing to disclose.

Parce que les mutations de la lignée germinale de la sonde du gène BRCA2 à un risque accru de plusieurs types de cancer, notamment le cancer femelle et mâle du sein, du cancer de l&#39;ovaire, le cancer de la prostate, le cancer du pancréas et le mélanome 3,4, un certain nombre d&#39;études ont été entreprises pour comprendre la fonction biologique de la protéine BRCA2. La plupart de ces études sont d&#39;origine génétique basée principalement en raison de difficultés techniques dans l...

Le gène BRCA2 (BReast susceptibilité au cancer gène-2) code pour une protéine suppresseur de tumeur qui joue un rôle crucial dans la réparation des cassures double-brin par régulation de la fonction de l&#39;enzyme recombinase Rad51 1. BRCA2 a également été impliquée dans la modulation de la transcription dans la cellule et le contrôle du cycle 2. Les mutations germinales dans le gène BRCA2 induisent un mode autosomique dominant la susceptibilité au cancer du sein et le cancer de l&#39;ovaire chez les femmes et le cancer de la prostate chez les hommes, ainsi que la prédisposition à d&#39;autres types de cancer 3,4. Cependant, en dépit de l&#39;augmentation du risque dans le développement de cancers, des mutations de BRCA2 qui suppriment ou réduisent fonction de BRCA2 peut rendre les cellules cancéreuses plus vulnérables aux agents chimiothérapeutiques qui causent des dommages de l&#39;ADN 5-7. Cancers sporadiques présentent un taux de mutations BRCA2 (&lt;3%) bien que des niveaux réduits de protéine BRCA2 ont été détectés dans certains types de cancers faible, ce qui suggère que la protéine BRCA2peut être perdue lors de la tumorigenèse dans les cancers sporadiques à travers des mécanismes non-mutation-dépendantes 7,8. Ainsi, il est important de bien comprendre les fonctions de BRCA2 dans le cadre de la biologie du cancer ainsi que dans d&#39;autres milieux biologiques. Un outil puissant utilisé pour identifier de nouvelles fonctions d&#39;un gène dans des cellules de mammifères est de réduire au silence l&#39;expression. A titre d&#39;exemple, réduire au silence l&#39;expression de BRCA2 dans une variété de cellules epitheliales normales a récemment conduit à l&#39;identification d&#39;une nouvelle fonction de BRCA2 en tant que régulateur de la résistance de anoikis, une étape importante lors de l&#39;acquisition de cellule cancéreuse capacité invasive et métastatique 9. Gene silencing peut être réalisé en introduisant dans les cellules soit faible interférence (si) ARN court en épingle à cheveux ou (sh) des molécules d&#39;ARN ciblant le gène spécifique. La puissance élevée de siRNA et sa facilité d&#39;utilisation en font un outil privilégié pour faire taire des expériences visant à acquérir de nouvelles connaissances sur les processus biologiques critiques, une pour identifier de nouvelles cibles thérapeutiques. Cependant, knockdown efficace de l&#39;expression génique peut ne pas être facilement réalisable pour tous les gènes et peut être très variable en fonction du type de cellule. Parce qu&#39;une diminution des niveaux de protéines intracellulaires est le phénotype le plus pertinent à l&#39;étude, il est crucial de quantifier l&#39;inactivation génique analyse par immunotransfert du produit de gène. Avec cet égard, la protéine BRCA2 présente une autre difficulté: être d&#39;une grande protéine (environ 390 kDa), des difficultés techniques existent pour l&#39;analyse biochimique classique, y compris immunotransfert. Nous rapportons ici un protocole optimisé pour une inactivation efficace de BRCA2 dans des lignées cellulaires epitheliales humaines et pour la détection rapide et efficace de la protéine BRCA2 knockdown analyse par immunotransfert.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BRCA2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-003462-00</td>
			<td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td>
		</tr>
		<tr>
			<td>Ctrl siRNA</td>
			<td>Dharmacon</td>
			<td>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>OPTI-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Medium for transfection procedure</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td>
			<td>Life Technologies</td>
			<td>EA0378</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample buffer&#160;</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing agent (10x)</td>
			<td>Life Technologies</td>
			<td>NP0004</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>Life Technologies</td>
			<td>NP0005</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>HiMark Pre-stained protein standard</td>
			<td>Life Technologies</td>
			<td>LC5699</td>
			<td>Prestained marker for gel electrophoresis</td>
		</tr>
		<tr>
			<td>XCell SureLoc Mini-Cell&#160; System</td>
			<td>Life Technologies</td>
			<td>EI0002</td>
			<td>Equipment for protein electrophoresis/transfer</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running Buffer</td>
			<td>Life Technologies</td>
			<td>LA0041</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Life Technologies</td>
			<td>NP0006-1</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>BRCA2 H-300 rabbit polyclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8326</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Beta-tubulin monoclonal antibody</td>
			<td>Sigma Aldrich</td>
			<td>T4026-100UL</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Skim Milk powder</td>
			<td>Sigma Aldrich</td>
			<td>70166</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td>
			<td>Pierce Thermo Scientific</td>
			<td>34080/34096</td>
			<td>Chemiluminescence system</td>
		</tr>
		<tr>
			<td>PNT1A cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>95012614</td>
			<td>PNT1A human, normal prostate epithelium immortalized with SV40</td>
		</tr>
		<tr>
			<td>Nthy cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>90011609</td>
			<td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit I (MTT)</td>
			<td>Roche</td>
			<td>11465007001</td>
			<td>Kit for cell proliferation assays</td>
		</tr>
	</tbody>
</table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

Silence de la protéine suppresseur de tumeur BRCA2 et sa détection par des analyses biochimiques classiques représentent un grand défi technique en raison de la grande taille de la protéine BRCA2 humain (environ 390 kDa). Nous rapportons modifications des protocoles standard de transfection et de réduire au silence immunoblotting siARN BRCA2 humain endogène et détecter la protéine BRCA2, respectivement, dans des lignées cellulaires epitheliales humaines. Les étapes clés comprennent une forte siRNA transfection ratio de réactif et de deux rondes subséquentes de transfection de siRNA dans la même expérience. L&#39;utilisation de ces et d&#39;autres modifications au protocole standard, nous réalisons systématiquement plus de 70% le silençage du gène BRCA2 humain tel que jugé par une analyse immunoblot avec des anticorps anti-BRCA2. En outre, la dénaturation des lysats de cellules à 55 ° C au lieu de la classique de 70 à 100 ° C et d&#39;autres techniques d&#39;optimisation de la procédure d&#39;immuno-empreinte permet la détection de la protéine BRCA2 intact même when très faibles quantités de matière de départ sont disponibles ou lorsque les niveaux d&#39;expression de protéine BRCA2 sont très faibles. Silençage efficace de BRCA2 dans les cellules humaines propose une stratégie utile pour perturber la fonction de BRCA2 dans les cellules avec BRCA2 intacte, y compris les cellules tumorales, à examiner de nouvelles voies moléculaires et les fonctions cellulaires qui peuvent être affectés par des mutations de BRCA2 pathogènes dans les tumeurs. Adaptation de ce protocole pour le silençage et l&#39;analyse d&#39;autres protéines «grands» comme BRCA2 efficace devrait être facilement réalisable.

Avant de procéder à un dosage biologique / biochimique pour examiner l&#39;effet de BRCA2 silence sur les fonctions des cellules, la première étape consiste à vérifier la spécificité de l&#39;inactivation à l&#39;aide d&#39;un ARNsi crypté (non-ciblage siRNA) côte à côte avec BRCA2 siRNA (figure 1A ). Il est important d&#39;effectuer un deuxième tour de transfection de siRNA avec un ratio élevé siRNA / transfection pour obtenir un rendement élevé de BRCA2 silencieux. ...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silence de<em&gt; BRCA2</em&gt; D&#39;identifier les fonctions biologiques de BRCA2 réglementés nouveaux dans les cellules humaines en culture

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K vues.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

Vidéo: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagerie en temps réel du noyau dense de vésicules cultures primaires neurones de l&#39;hippocampe

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Recherche

Biologie

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolement du ribosome Bound polypeptides naissants<em&gt; In vitro</em&gt; Pour identifier les sites de pause de translation le long d&#39;ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Un dosage quantitatif pour l&#39;étude de protéines: ADN Interactions, Découverte de transcription régulateurs de l&#39;expression des gènes, et identifier les agents anti-tumoraux

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualisation de réticulum endoplasmique sous-domaines dans les cellules cultivées

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Captation composé spectrométrie de masse - un outil puissant pour identifier de nouveaux c-di-GMP protéines effectrices

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA contrôle pour la ubiquitine et les régulateurs du système ubiquitine-comme des voies biologiques dans des cellules de mammifères en culture

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Mesure de l&#39;absorption de glucose et réponse à la stimulation de l&#39;insuline dans<em&gt; In Vitro</em&gt; Myotubes primaires humains cultivés

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...