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. Preparare le soluzioni di siRNA <ol><li> Risospendere 5 nmol di strapazzate (Ctrl) e 5 nmol di BRCA2 siRNA pellet secchi in 100 ml di acqua RNase-free (concentrazione siRNA finale: 50 micron). Questo è lo stock siRNA e deve essere conservato a -20 ° C in 10 microlitri aliquote. </li><li> Prendete una aliquota di 10 ml dal magazzino siRNA e aggiungere 40 ml di acqua RNase-free per ottenere la soluzione di lavoro 10 micron siRNA. Questa diluizione deve essere conservata a -20 ° C fino al momento dell&#39;uso. NOTA: La soluzione di lavoro siRNA non deve essere sottoposto a congelamento / scongelamento più di 3 volte. </li></ol> 2. Semina di epiteliali umane linee cellulari <ol><li> Rimuovere il mezzo di crescita di linee cellulari epiteliali umane che crescono come monostrati di cellule in piastre di coltura di tessuti in un incubatore umidificato a 37 ° C, 5% CO 2. </li><li> Lavare le cellule nella piastra una volta con PBS RT (10 ml di PBS per una piastra di 100 mm). </li><li> Per staccare le cellule dalla piastra,aggiungere 2 ml di 0,25% tripsina / EDTA soluzione 0,53 mM e incubare 3-5 min a seconda del tipo di cellula. </li><li> Prima di raccogliere le cellule staccate, neutralizzare tripsina aggiungendo 2 ml di terreno di coltura completo contenente siero bovino fetale al 10%. </li><li> Trasferire le cellule in una provetta da 15 ml. </li><li> Centrifugare le cellule a 320-330 xg per 3 min a 4 ° C in un rotore oscillante secchio. </li><li> Risospendere il pellet di cellule in 5 ml di free-antibiotici medie. </li><li> Contare le cellule sotto il microscopio in una camera di Burker o utilizzando un sistema di conteggio automatico secondo le istruzioni del produttore. </li><li> Seed le cellule in 6 pozzetti per essere circa l&#39;80% confluenti dopo 24 ore (0,5-1 x10 6 celle in 2 ml di terreno per ogni bene). Numero ottimale di cellule per essere seminati possono variare a seconda delle linee cellulari specifiche e il tasso di crescita. NOTA: Non aggiungere antibiotici nel mezzo a questo punto, perché gli antibiotici interferiscono negativamente con l&#39;efficienza di trasferction. È molto importante che le cellule sono a passaggi su coltura basse (&lt;10) per ottenere un&#39;elevata efficienza di BRCA2 siRNA trasfezione. </li></ol> 3. Le cellule trasfezione con siRNA <ol><li> Ventiquattro ore dopo la placcatura le cellule, procedere con il primo turno di trasfezione. <ol><li> Diluire 6 ml di base lipidica 10 siRNA specifico reagente di trasfezione in 130 ml di mezzo di siero ridotto. </li><li> Diluire 3 ml di 10 micron siRNA (30 pmol) in 130 ml di mezzo di siero ridotto. </li><li> Unire il siRNA diluito con il reagente di trasfezione diluito e incubare per 5-10 minuti a temperatura ambiente. </li><li> Durante l&#39;incubazione, rimuovere il terreno dalle cellule e aggiungere 2 ml di mezzo fresco pre-riscaldato a 37 ° C (senza antibiotici). </li><li> Aggiungere 250 l di siRNA-trasfezione complessi reagenti alle cellule (l&#39;importo è per un singolo pozzo di un 6-pozzetti). </li><li> Incubare le cellule in un incubatore umidificato a 3776; C con 5% di CO 2. </li></ol></li><li> Dopo 24 ore, procedere con il secondo turno di trasfezione ripetendo i punti 3.1.1-3.1.5 con la seguente modifica di passo 3.1.2: diluire 5 ml di 10 micron siRNA (50 pmoli) in 130 ml di mezzo di siero ridotto. <ol><li> Incubare le cellule per 1-2 giorni a 37 ° C prima dell&#39;analisi. NOTA: Due turni di trasfezioni e l&#39;uso di un rapporto di reagenti alto siRNA / trasfezione al secondo turno sono essenziali per ottenere un&#39;elevata efficienza di silenziamento BRCA2. </li></ol></li></ol> 4. cellule Lisare per l&#39;analisi immunoblotting <ol><li> Preparare tampone di lisi fresca con PBS (pH 7.4) contenente 1% di non ionico, non denaturante detergente ottilfenossipolietossietanolo, 5 mM sodio pirofosfato, 1 mM orthovanadate di sodio, fluoruro di sodio 10 mM, fluoruro phenylmethylsulfonyl 2 mM, 10 mg / ml leupeptina, 10 ug / ml aprotinina. Keep it sul ghiaccio. </li><li> Rimuovere il supporto da parte delle cellule e wcenere una volta con PBS freddo (2 ml / pozzetto) nella piastra. Per evitare qualsiasi riporto PBS, rimuoverlo completamente dalla piastra. </li><li> Aggiungere 200 ml di tampone di lisi a ciascun pozzetto. NOTA: Se le cellule devono essere utilizzati anche per altri saggi, trypsinize le cellule come descritto al punto 2.1-2.6 e risospendere le cellule in appropriato tampone / supporto secondo l&#39;applicazione da usare. </li><li> Ruotare delicatamente la piastra per distribuire uniformemente il tampone di lisi e incubare per 15 min a 4 ° C su un rotatore. </li><li> Raccogliere il lisato cellulare in una provetta su ghiaccio con un raschietto cellulare. </li><li> Centrifugare a 17.900 xg per 25 min a 4 ° C e raccogliere il surnatante in una nuova provetta. </li><li> Misurare la concentrazione proteica utilizzando un saggio proteico disponibile in commercio secondo le istruzioni del produttore. </li></ol> 5. BRCA2 analisi immunoblotting <ol><li> Preparare il tampone di corsa diluendo 50 ml di 20x Tris-acetato SDS esecuzione bUffer (Tricine 50 mM, 50 mM Tris Base, 0,1% SDS, pH 8.24) con 950 ml di acqua distillata. </li><li> Preparare il campione come segue: aggiungere 15 ug di lisato cellulare totale, 5 ml di tampone campione 4x contenenti litio dodecil solfato a pH 8,4, 2 ml di agente riducente 10x campione (0.5 M DTT), e tampone di lisi a un volume finale di 20 ul. NOTA: Questo protocollo consente il rilevamento di BRCA2 in linee cellulari normali anche quando si utilizza una minore quantità di proteine ​​totali (fino a 5 mg). </li><li> Denaturare i campioni a 55 ° C per 10 min. NOTA: E &#39;fondamentale utilizzare 55 ° C. Dal momento che la proteina BRCA2 è termosensibile 11, le temperature più elevate denaturanti provocano la perdita costante di intatta tutta lunghezza (390 kDa) proteina BRCA2. </li><li> Impostare la camera elettroforetico secondo le istruzioni del produttore. </li><li> Caricare i campioni e 10 ml di alto peso molecolare pre-tinto standard di proteine ​​su un gel precasted (Tris-acetato 3-8%) e correre a 120 V per abfuori 2 ore 12. Fermare la corsa elettroforetica prima del 55 kDa indicatore blu lascia il gel precasted. </li><li> Preparare il buffer di trasferimento con 50 ml di 20x tampone di trasferimento, 100 ml di metanolo 100% e 850 ml di acqua. </li><li> Attiva membrana PVDF (0,45 micron dimensione dei pori) immergendolo in metanolo 100% per 5 minuti, risciacquare con acqua distillata ed equilibrare in tampone di trasferimento almeno per 5 min. Immergere le spugne del dispositivo di trasferimento in tampone di trasferimento a 4 ° C fino al momento dell&#39;uso. </li><li> Quando la corsa elettroforetica è finita, montare il sandwich nella pila di trasferimento nel seguente ordine (bottom-up): tre spugne saturi nel buffer di trasferimento, un foglio di carta 3MM grado saturo in buffer di trasferimento, il gel, membrane PVDF attivato, uno 3MM foglio di carta di grado saturi in buffer di trasferimento, tre spugne saturo in buffer di trasferimento 13,14. Posizionare la pila nelle proteine ​​apparecchi e di trasferimento a 350 mA per 4 ore a 4 ° C o 180 mA notte a 4 ° C. NOTA: Essendo BRCA2 una grande proteina, è fondamentale per estendere il tempo di trasferimento da 1 hr (come suggerito dal produttore e la maggior parte dei protocolli di trasferimento) per 4 ore o durante la notte per garantire la completa trasferimento di proteine ​​ad alto peso molecolare. In aggiunta, a causa del tempo di trasferimento lungo, è indispensabile effettuare il blotting in una camera fredda a 4 ° C per ridurre al minimo il surriscaldamento e facilitare smontaggio sandwich. Sistemi di trasferimento semi-dry non sono buone per il trasferimento di grandi proteine. </li><li> Dopo il trasferimento, lavare la membrana con TBS, bloccarlo per 1 ora a RT in TBS-Tween 0,25% (TBS-T) contenente latte in polvere 5% nonfat e sonda notte a 4 ° C con 30 microlitri anti-BRCA2 anticorpo policlonale di coniglio ( 200 mcg / ml), diluito in 9 ml TBS-T + 5% latte scremato secca [1: 300 (v / v)] diluizione. </li><li> Lavare la membrana tre volte (10 min ciascuno) con TBS-T, allora il filtro incubare per 1 ora con 1 ml di rafano anti-coniglio anticorpo secondario coniugato con perossidasi diluito in 10 ml di TBS-T + 5% senza grassi latte in polvere. Successivamente, lavare la membrana per tre volte (10 min ciascuno) con TBS-T. </li><li> Eseguire immunolocalizzazione di ECL per rivelare la proteina BRCA2 con chemiluminescenza (ECL) Western Blotting Detection Reagent, seguendo le istruzioni del produttore. Visualizza bande immunochemiluminescenti ad alta risoluzione su pellicole ECL. </li><li> Eseguire immunolocalizzazione con un anticorpo per un gene housekeeping, come β-tubulina (55 kDa), sullo stesso filtro come controllo di caricamento utilizzando un anticorpo monoclonale di β-tubulina diluito a 1: 1.000 (10 ml di anticorpo anti-tubulina in 10 ml TBS-T latte in polvere senza grassi + 5%) per 1 ora a temperatura ambiente. Lavare e incubare anticorpo secondario come descritto per BRCA2 (5,10-5,11), fatta eccezione per l&#39;utilizzo di perossidasi di rafano-coniugato anti-mouse invece di anticorpo secondario anti-coniglio. </li><li> Acquisire un&#39;immagine digitale dei film impressi con bande immunochemiluminescenti e analizzare l&#39;intensità della banda da immagine dedicato software di analisi (per esempio., ImageJ). </li></ol>

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The authors have nothing to disclose.

A causa mutazioni germinali del gene BRCA2 piombo ad un aumentato rischio di diversi tipi di cancro, tra cui il cancro al seno maschile e femminile, il cancro ovarico, il cancro alla prostata, cancro al pancreas, e il melanoma 3,4, sono state avviate una serie di studi per comprendere la funzione biologica della proteina BRCA2. La maggior parte di questi studi sono genetiche a base principalmente a causa di difficoltà tecniche analizzando una proteina gigante come BRCA2. Il metodo descritto qui di s...

Il BRCA2 (Breast Cancer suscettibilità gene-2) gene codifica per una proteina soppressore del tumore che gioca un ruolo cruciale nella riparazione del DNA rotture del doppio filamento regolando la funzione dell&#39;enzima recombinase Rad51 1. BRCA2 è stato anche coinvolto nella modulazione della trascrizione e nel controllo del ciclo cellulare 2. Mutazioni germinali nel gene BRCA2 inducono un autosomica dominante suscettibilità cancro al seno e alle ovaie nelle donne e il cancro alla prostata negli uomini, nonché la predisposizione di altri tipi di cancro 3,4. Tuttavia, nonostante l&#39;aumento del rischio di sviluppare il cancro, mutazioni BRCA2 che sopprimono o riducono la funzionalità BRCA2 possono rendere le cellule tumorali più vulnerabili agli agenti chemioterapici che causano danni al DNA 5-7. Tumori sporadici presentano un basso tasso di mutazioni BRCA2 (&lt;3%) anche se sono stati rilevati livelli ridotti di proteina BRCA2 in alcuni tipi di cancro, suggerendo che la proteina BRCA2può essere perso durante la tumorigenesi nei tumori sporadici attraverso meccanismi non mutazionale dipendenti 7,8. Pertanto, è importante comprendere appieno funzioni BRCA2 nel contesto della biologia cancro e in altri contesti biologici. Uno strumento potente usato per identificare nuove funzioni di un gene in cellule di mammifero è di mettere a tacere la sua espressione. Come esempio, il silenziamento dell&#39;espressione BRCA2 in una varietà di cellule epiteliali normali ha recentemente portato all&#39;identificazione di una nuova funzione di BRCA2 come regolatore di anoikis resistenza, un passo importante durante l&#39;acquisizione di cellule di cancro invasivo e metastatico capacità 9. Gene silenziamento può essere raggiunto con l&#39;introduzione nelle cellule o small interfering (si) RNA o brevi tornanti (sh) molecole di RNA rivolti al gene specifico. L&#39;elevata potenza di siRNA e la sua facilità d&#39;uso ne fanno lo strumento preferenziale per tacere esperimenti volti ad acquisire nuove conoscenze sui processi biologici critici unnd di identificare nuovi bersagli terapeutici. Tuttavia, l&#39;abbattimento efficiente di espressione genica può non essere facilmente raggiungibile per tutti i geni e può essere molto variabile a seconda del tipo di cellula. Poiché una diminuzione dei livelli di proteine ​​intracellulari è il fenotipo più rilevante in esame, è fondamentale quantificare silenziamento genico mediante immunoblotting analisi del prodotto del gene. Con questo proposito, la proteina BRCA2 presenta un&#39;ulteriore sfida: essere una grande proteina (circa 390 kDa), esistono difficoltà tecniche per l&#39;analisi biochimica convenzionale, compresi immunoblotting. Riportiamo qui un protocollo ottimizzato per tacere efficiente di BRCA2 in linee cellulari epiteliali umane e per la rilevazione rapida e positiva di proteine ​​BRCA2 knockdown mediante immunoblotting analisi.

<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

Silenziamento della proteina BRCA2 soppressore del tumore e la sua individuazione da analisi biochimiche convenzionali rappresentano una grande sfida tecnica a causa delle grandi dimensioni della proteina BRCA2 umana (circa 390 kDa). Riportiamo modifiche di protocolli di trasfezione e immunoblotting siRNA standard per tacere BRCA2 umano e rilevare proteina BRCA2 endogena, rispettivamente, nelle linee di cellule epiteliali umane. Passaggi chiave includono un alto siRNA rapporto reagente di trasfezione e due successivi turni di siRNA trasfezione all&#39;interno dello stesso esperimento. Utilizzando queste e altre modifiche al protocollo standard che costantemente raggiungere oltre il 70% silenziamento del gene BRCA2 umano come giudicato da immunoblotting con anticorpi anti-BRCA2. Inoltre, denaturazione dei lisati cellulari a 55 ° C invece che nel tradizionale 70-100 ° C e altre ottimizzazioni tecniche della procedura immunoblotting consentono di individuare proteina BRCA2 intatta anche when molto basse quantità di materiale di partenza sono disponibili o quando i livelli di espressione della proteina BRCA2 sono molto basse. Silenziamento efficiente di BRCA2 nelle cellule umane offre una valida strategia per interrompere la funzione BRCA2 in cellule con BRCA2 intatte, comprese le cellule tumorali, per esaminare nuovi percorsi molecolari e funzioni cellulari che possono essere interessati da mutazioni BRCA2 patogeni nei tumori. L&#39;adattamento di questo protocollo per tacere e l&#39;analisi di altre proteine ​​&quot;grandi&quot; come BRCA2 efficiente dovrebbe essere facilmente realizzabile.

Prima di procedere con un saggio biologico / biochimico per esaminare l&#39;effetto di silenziamento BRCA2 sulle funzioni cellulari, il primo passo è per confermare la specificità del silenziamento utilizzando un siRNA criptato (non-targeting siRNA) fianco a fianco con BRCA2 siRNA (Figura 1A ). E &#39;importante eseguire una seconda tornata di siRNA trasfezione con un elevato rapporto siRNA / trasfezione per ottenere un&#39;elevata efficienza di silenziamento BRCA2. In effetti, l&#39...

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.

Silencing di<em&gt; BRCA2</em&gt; Per identificare le funzioni biologiche Novel BRCA2-regolamentato coltivate cellule umane

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 Views.  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. 

Video: 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.

Immagini dal vivo di Dense-core Vescicole nella Primaria neuroni coltivati ​​ippocampale

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

Ricerca

Biologia

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.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

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 quantitativo test per studiare proteine: DNA interazioni, Discover trascrizionali regolatori dell&#39;espressione genica, e identificare nuovi agenti anti-tumorali

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.

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

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.

Capture spettrometria di massa Compound - un potente strumento per identificare nuovi c-di-GMP effettori Proteine

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 screening per identificare ubiquitina e ubiquitina-come regolatori del sistema di percorsi biologici in cellule di mammifero coltivate

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.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

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.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

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.

Misurazione e risposta alla somministrazione di glucosio alla stimolazione dell&#39;insulina in<em&gt; In vitro</em&gt; Myotubes primario umano coltivato

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