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. Prepare siRNA solutions
<ol>
	<li>Resuspend 5 nmol of scrambled (Ctrl) and 5 nmol of BRCA2 siRNA dried pellets in 100 &#181;l of RNase-free water (final siRNA concentration: 50 &#181;M). This is the siRNA stock and must be stored at -20 &#176;C in 10 &#181;l aliquots.</li>
	<li>Take an aliquot of 10 &#181;l from the siRNA stock and add 40 &#181;l of RNase-free water to obtain the 10 &#181;M siRNA working solution. This dilution must be stored at -20 &#176;C until use. 
	NOTE: The siRNA working solution should not undergo freezing/thawing more than 3 times.</li>
</ol>
2. Seeding of Human Epithelial Cell Lines
<ol>
	<li>Remove the growth medium from human epithelial cell lines growing as cell monolayers in tissue culture plates in a humidified incubator at 37 &#176;C, 5% CO2.</li>
	<li>Wash the cells in the plate once with RT PBS (10 ml of PBS for a 100 mm plate).</li>
	<li>To detach the cells from the plate, add 2 ml of 0.25% trypsin/0.53 mM EDTA solution and incubate 3-5 min depending on the cell type.</li>
	<li>Before collecting the detached cells, neutralize trypsin by adding 2 ml of complete growth medium containing 10% fetal bovine serum.</li>
	<li>Transfer the cells to a 15 ml tube.</li>
	<li>Centrifuge the cells at 320-330 x g for 3 min at 4 &#176;C in a swinging bucket rotor.</li>
	<li>Resuspend the cell pellets in 5 ml of antibiotics-free medium.</li>
	<li>Count the cells under the microscope in a Burker chamber or by using an automated counting system according to the manufacturer&#8217;s instructions.</li>
	<li>Seed the cells in 6-well plates to be about 80% confluent after 24 hr (0.5-1 x106 cells in 2 ml of medium for each well). Optimal cell number to be seeded may vary depending on the specific cell lines and the growth rate. 
	NOTE: Do not add antibiotics in the medium at this point because antibiotics will negatively interfere with the efficiency of transfection. It is very important that the cells are at low culture passages (&#60; 10) to achieve high efficiency of BRCA2 siRNA transfection.</li>
</ol>
3. Transfect cells with siRNA
<ol>
	<li>Twenty-four hours after plating the cells, proceed with the first round of transfection.
	<ol>
		<li>Dilute 6 &#181;l of lipid-based 10 siRNA specific transfection reagent in 130 &#181;l of reduced serum medium.</li>
		<li>Dilute 3 &#181;l of 10 &#181;M siRNA (30 pmol) in 130 &#181;l of reduced serum medium.</li>
		<li>Combine the diluted siRNA with the diluted transfection reagent and incubate for 5-10 min at RT.</li>
		<li>During the incubation, remove the medium from the cells and add 2 ml of fresh medium pre-warmed at 37 &#176;C (without antibiotics).</li>
		<li>Add 250 &#181;l of the siRNA-transfection reagent complexes to the cells (this amount is for a single well of a 6-well plate).</li>
		<li>Incubate the cells in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>After 24 hr, proceed with the second round of transfection by repeating steps 3.1.1-3.1.5 with the following modification for step 3.1.2: dilute 5 &#181;l of 10 &#181;M siRNA (50 pmol) in 130 &#181;l of reduced serum medium.
	<ol>
		<li>Incubate the cells for 1-2 days at 37 &#176;C before analysis. 
		NOTE: Two rounds of transfections and use of a high siRNA/transfection reagent ratio in the second round are essential to get high efficiency of BRCA2 silencing.</li>
	</ol>
	</li>
</ol>
4. Lyse cells for immunoblotting analysis
<ol>
	<li>&#160;Prepare fresh lysis buffer with PBS (pH 7.4) containing 1% of the nonionic, non-denaturing detergent octylphenoxypolyethoxyethanol, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, 10 &#181;g/ml leupeptin, 10 &#181;g/ml aprotinin. Keep it on ice.</li>
	<li>Remove the medium from the cells and wash them once with cold PBS (2 ml/well) in the plate. To prevent any PBS carryover, completely remove it from the plate.</li>
	<li>Add 200 &#181;l of lysis buffer to each well. 
	NOTE: If the cells have to be used also for other assays, trypsinize the cells as described in step 2.1-2.6 and resuspend the cells in appropriate buffer/medium according to the application to be used.</li>
	<li>Gently rotate the plate to uniformly distribute the lysis buffer and incubate for 15 min at 4 &#176;C on a rotator.</li>
	<li>Collect the cell lysate in a microcentrifuge tube on ice using a cell scraper.</li>
	<li>Centrifuge at 17,900 x g for 25 min at 4 &#176;C and collect the supernatant in a new tube.</li>
	<li>Measure the protein concentration using a commercially available protein assay according to the manufacturer&#39;s instructions.</li>
</ol>
5. BRCA2 immunoblotting analysis
<ol>
	<li>Prepare the running buffer by diluting 50 ml of 20x Tris-Acetate SDS running buffer (50 mM Tricine, 50 mM Tris Base, 0.1% SDS, pH 8.24) with 950 ml of distilled water.</li>
	<li>Prepare the sample as follows: add 15 &#181;g of total cell lysate, 5 &#181;l of 4x sample buffer containing lithium dodecyl sulfate at pH 8.4, 2 &#181;l of 10x sample reducing agent (0.5 M DTT), and lysis buffer to a final volume of 20 &#181;l. 
	NOTE: This protocol allows detection of BRCA2 in normal cell lines also when using a lower amount of total proteins (down to 5 &#181;g).</li>
	<li>Denature samples at 55 &#176;C for 10 min. 
	NOTE: It is crucial to use 55 &#176;C. Since BRCA2 protein is thermosensitive 11, higher denaturing temperatures result in consistent loss of intact full-length (390 kDa) BRCA2 protein.</li>
	<li>Set up the electrophoretic chamber according to the manufacturer&#39;s instructions.</li>
	<li>Load the samples and 10 &#181;l of high-molecular weight pre-stained protein standard on a precasted gel (Tris-Acetate 3-8%) and run at 120 V for about 2 hr 12. Stop the electrophoretic run before the 55 kDa blue marker leaves the precasted gel.</li>
	<li>Prepare the transfer buffer with 50 ml of 20x transfer buffer, 100 ml of 100% methanol and 850 ml water.</li>
	<li>Activate PVDF membrane (0.45 &#181;m pore size) by soaking in 100% methanol for 5 min, rinse with distilled water and equilibrate in transfer buffer at least for 5 min. Soak the sponges of the transfer apparatus in Transfer buffer at 4 &#176;C until use.</li>
	<li>When the electrophoretic run is over, assemble the sandwich in the transfer stack in the following order (bottom-up): three sponges saturated in transfer buffer, one 3MM grade paper sheet saturated in transfer buffer, the gel, activated PVDF membrane, one 3MM grade paper sheet saturated in transfer buffer, three sponges saturated in transfer buffer 13,14. Place the stack into the apparatus and transfer proteins at 350 mA for 4 hr at 4 &#176;C or at 180 mA overnight at 4 &#176;C. 
	NOTE: Being BRCA2 a very large protein, it is crucial to extend the transfer time from 1 hr (as suggested by the manufacturer and most transfer protocols) to 4 hr or overnight to ensure complete transfer of high-molecular weight proteins. In addition, due to the long transfer time, it is essential to perform the blotting in a cold room at 4 &#176;C to minimize overheating and facilitate sandwich disassembling. Semi-dry transfer systems are not good for transferring large proteins.</li>
	<li>After transfer, wash the membrane with TBS, block it for 1 hr at RT in TBS-Tween 0.25% (TBS-T) containing 5% nonfat dry milk and probe overnight at 4 &#176;C with 30 &#956;l anti-BRCA2 rabbit polyclonal antibody (200 &#956;g/ml), diluted in 9 ml TBS-T + 5% nonfat dry milk [1:300 (v/v) dilution].</li>
	<li>Wash the membrane three times (10 min each) with TBS-T, then incubate the filter for 1 hr with 1 &#956;l of horseradish peroxidase-conjugated anti-rabbit secondary antibody diluted in 10 ml of TBS-T + 5% nonfat dry milk. Thereafter, wash the membrane three times (10 min each) with TBS-T.</li>
	<li>Perform immunodetection by ECL to reveal BRCA2 protein using enhanced chemiluminescence (ECL) Western Blotting Detection Reagent, following the manufacturer&#39;s instructions. Visualize immunochemiluminescent bands on high resolution ECL films.</li>
	<li>Perform immunodetection with an antibody for a housekeeping gene, like &#946;-tubulin (55 kDa), on the same filter as loading control by using a monoclonal antibody to &#946;-tubulin diluted at 1:1,000 (10 &#181;l of anti-tubulin antibody in 10 ml TBS-T+5% non-fat dry milk) for 1 hr at RT. Wash and incubate secondary antibody as described for BRCA2 (5.10-5.11), except for the use of horseradish peroxidase-conjugated anti-mouse instead of anti-rabbit secondary antibody.</li>
	<li>Acquire a digital image of the films impressed with immunochemiluminescent bands and analyze the band intensity by dedicated image analysis software (e.g., 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.

Because germline mutations of the BRCA2 gene lead to increased risk of several cancer types, including female and male breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and melanoma 3,4, a number of studies have been undertaken to understand the biological function of the BRCA2 protein. Most of these studies are genetic-based mainly due to technical difficulties in analyzing a giant protein like BRCA2. The method described here for silencing and analyzing BRCA2 protein expression prov...

The BRCA2 (BReast CAncer susceptibility gene-2) gene encodes for a tumor suppressor protein that plays a crucial role in repairing DNA double-strand breaks by regulating the function of the recombinase enzyme Rad51 1. BRCA2 has also been implicated in the modulation of transcription and in cell cycle control 2. Germline mutations in the BRCA2 gene induce an autosomal dominant susceptibility to breast and ovarian cancer in women and prostate cancer in men, as well as predisposition to other cancer types 3,4. However, despite the increased risk in developing cancer, BRCA2 mutations that suppress or reduce BRCA2 function may render cancer cells more vulnerable to chemotherapeutic agents that cause DNA damage 5-7. Sporadic cancers exhibit a low rate of BRCA2 mutations (&#60; 3%) though reduced levels of BRCA2 protein have been detected in some cancer types, suggesting that BRCA2 protein may be lost during tumorigenesis in sporadic cancers through non-mutational-dependent mechanisms 7,8. Thus, it is important to fully understand BRCA2 functions in the context of cancer biology as well as in other biological settings.
A powerful tool used to identify novel functions of a gene in mammalian cells is to silence its expression. As an example, silencing BRCA2 expression in a variety of normal epithelial cells has recently led to the identification of a novel function of BRCA2 as regulator of anoikis resistance, an important step during acquisition of cancer cell invasive and metastatic ability 9. Gene silencing can be achieved by introducing in the cells either small interfering (si) RNA or short hairpin (sh) RNA molecules targeting the specific gene. The high potency of siRNA and its ease of use make it the preferential tool for silencing experiments aimed at gaining new insights into critical biological processes and to identify novel therapeutic targets. However, efficient knockdown of gene expression may not be easily achievable for all genes and may be highly variable depending on the cell type. Because a decrease in intracellular protein levels is the most relevant phenotype under investigation, it is crucial to quantify gene silencing by immunoblotting analysis of the gene product. With this respect, the BRCA2 protein presents a further challenge: being a large protein (approximately 390 kDa), technical difficulties do exist for conventional biochemical analysis, including immunoblotting.
We report here a protocol optimized for efficient silencing of BRCA2 in human epithelial cell lines and for rapid and successful detection of BRCA2 protein knockdown by immunoblotting analysis.

<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

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Before proceeding with a biological/biochemical assay to examine the effect of BRCA2 silencing on cell functions, the first step is to confirm the specificity of the silencing by using a scrambled siRNA (non-targeting siRNA) side by side with BRCA2 siRNA (Figure 1A). It is important to perform a second round of siRNA transfection with a high siRNA/transfection ratio to get a high efficiency of BRCA2 silencing. Indeed, performing only one round of siRNA transfection or using a lower siRN...

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

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

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. 

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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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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