Name: stable knockdown of genes encoding extracellular matrix proteins in the c2c12 myoblast cell line using small-hairpin (sh)rna
Uploaded: 2020-02-12T13:30:00
Description: Watch this Scientific Journal Video about Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA at JoVE.com

D.H. is supported by the National Institutes of Health (National Institute for Arthritis and Musculoskeletal and Skin Diseases, NIAMS, grant number AR070748) and seed funding from the Leni &#38; Peter W. May Department of Orthopedics, Icahn School of Medicine at Mt. Sinai.

1. Preparing the shRNA Plasmid DNA from Escherichia coli
<ol>
	<li>Generation of clonal bacterial colonies carrying the shRNA plasmids
	<ol>
		<li>Obtain glycerol stocks of E. coli carrying target-specific shRNA plasmids and a control plasmid from commercial sources (Table of Materials). 
		NOTE: Three different shRNA plasmids were used, targeting different regions of the murine Adamtsl2 mRNA. One shRNA was selected to target the 3&#39;-untranslated region (3&#39;UTR) of...

<ol>
	<li>Tanzer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+of+extracellular+matrix.">Current concepts of extracellular matrix.</a> Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. 11 (3), 326-331 (2006).</li><li>Hubmacher, D., Apte, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+the+extracellular+matrix:+novel+insights.">The biology of the extracellular matrix: novel insights.</a> Current Opinion in Rheumatology. 25 (1), 65-70 (2013).</li><li>Sakai, L. Y., Keene, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix:+The+driving+force+of+mammalian+diseases.">Extracellular matrix: The driving force of mammalian diseases.</a> Matrix Biology. 71-72, 1-9 (2018).</li><li>Le Goff, C., 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=ADAMTSL2+mutations+in+geleophysic+dysplasia+demonstrate+a+role+for+ADAMTS-like+proteins+in+TGF-beta+bioavailability+regulation.">ADAMTSL2 mutations in geleophysic dysplasia demonstrate a role for ADAMTS-like proteins in TGF-beta bioavailability regulation.</a> Nature Genetics. 40 (9), 1119-1123 (2008).</li><li>Dubail, J., Apte, S. 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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=ADAMTS-like+2+(ADAMTSL2)+is+a+secreted+glycoprotein+that+is+widely+expressed+during+mouse+embryogenesis+and+is+regulated+during+skeletal+myogenesis.">ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis.</a> Matrix Biology. 26 (6), 431-441 (2007).</li><li>Khodabukus, A., Baar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+serum+origin+on+tissue+engineered+skeletal+muscle+function.">The effect of serum origin on tissue engineered skeletal muscle function.</a> Journal of Cellular Biochemistry. 115 (12), 2198-2207 (2014).</li><li>Bajaj, P., 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=Patterning+the+differentiation+of+C2C12+skeletal+myoblasts.">Patterning the differentiation of C2C12 skeletal myoblasts.</a> Integrative Biology. 3 (9), 897-909 (2011).</li><li>Mashinchian, O., Pisconti, A., Le Moal, E., Bentzinger, C. 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S., 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=Conditions+that+promote+primary+human+skeletal+myoblast+culture+and+muscle+differentiation+in+vitro.">Conditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro.</a> American Journal of Physiology-Cell Physiology. 306 (4), 385-395 (2014).</li><li>Conejo, R., Valverde, A. M., Benito, M., Lorenzo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+produces+myogenesis+in+C2C12+myoblasts+by+induction+of+NF-kappaB+and+downregulation+of+AP-1+activities.">Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities.</a> Journal of Cellular Physiology. 186 (1), 82-94 (2001).</li><li>Dodds, E., Dunckley, M. G., Naujoks, K., Michaelis, U., Dickson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection+of+cultured+mouse+muscle+cells:+a+direct+comparison+of+Lipofectamine+and+DOSPER.">Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.</a> Gene Therapy. 5 (4), 542-551 (1998).</li><li>Balc&#305;, B., Din&#231;er, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+mouse-derived+C2C12+myoblasts+using+a+matrigel+basement+membrane+matrix.">Efficient transfection of mouse-derived C2C12 myoblasts using a matrigel basement membrane matrix.</a> Biotechnology Journal. 4 (7), 1042-1045 (2009).</li><li>Xia, D., 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=Overexpression+of+chemokine-like+factor+2+promotes+the+proliferation+and+survival+of+C2C12+skeletal+muscle+cells.">Overexpression of chemokine-like factor 2 promotes the proliferation and survival of C2C12 skeletal muscle cells.</a> Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1591 (1), 163-173 (2002).</li><li>Tapia, O., Gerace, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Nuclear+Lamina+Proteins+in+Myoblast+Differentiation+by+Functional+Complementation.">Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.</a> Methods in Molecular Biology. 1411, 177-194 (2016).</li><li>Yamano, S., Dai, J., Moursi, A. 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We describe here a protocol for the stable knockdown of ECM proteins in C2C12 myoblasts and for phenotypic analysis of the differentiation of C2C12 myoblasts into myotubes. Several factors determine the outcome of the experiment and need to be considered carefully. Maintaining C2C12 cells in the proliferating phase is a critical step to keep the C2C12 cells in the myoblast precursor state. Retaining the capability of C2C12 cells to consistently differentiate into myotubes depends on i) the passage number of the cells, ii...

Extracellular matrix (ECM) proteins provide structural support for all tissues, mediate cell-cell communication, and determine cell fate. The formation and dynamic remodeling of ECM is thus critical to maintain tissue and organ homeostasis1,2. Pathological variants in several genes coding for ECM proteins give rise to musculoskeletal disorders with phenotypes ranging from muscular dystrophies to pseudomouscular build3,4. For example, pathogenic variants in ADAMTSL2 cause the extremely rare musculoskeletal disorder geleophysic dysplasia, which presents with pseudomuscular build, i.e., an apparent increase in skeletal muscle mass5. Together with gene expression data in mouse and humans, this suggests a role for ADAMTSL2 in skeletal muscle development or homeostasis6,7.
The protocol that we describe here was developed to study the mechanism by which ADAMTSL2 modulates skeletal muscle development and/or homeostasis in a cell culture setting. We stably knocked down ADAMTSL2 in the murine C2C12 myoblast cell line. C2C12 myoblasts and their differentiation into myotubes is a well-described and widely used cell culture model for skeletal muscle differentiation and skeletal muscle bioengineering8,9. C2C12 cells go through distinct differentiation steps after serum withdrawal, resulting in the formation of multinucleated myotubes after 3&#8722;10 days in culture. These differentiation steps can be reliably monitored by measuring mRNA levels of distinct marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC). One advantage of generating stable gene knockdowns in C2C12 cells is that genes that are expressed at later stages of C2C12 differentiation can be targeted more efficiently, compared to transient knockdown achieved by small-interfering (si) RNA, which typically lasts for 5&#8722;7 days after transfection, and is influenced by the transfection efficiency. A second advantage of the protocol as described here is the relatively fast generation of batches of C2C12 knockdown cells using puromycin selection. Alternatives, such as CRISPR/Cas9-mediated gene knockout or the isolation of primary skeletal muscle cell precursors from human or target-gene deficient mice are technically more challenging or require the availability of patient muscle biopsies or target-gene deficient mice, respectively. However, similar to other cell culture based approaches, there are limitations in the use of C2C12 cells as model for skeletal muscle cell differentiation, such as the two-dimensional (2D) nature of the cell culture set-up and the lack of the in vivo microenvironment that is critical to maintain undifferentiated skeletal muscle precursor cells10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Fisher Chemical</td>
			<td>191784</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Bioreagents</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1760-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter Countesse II</td>
			<td>Invitrogen</td>
			<td>A27977</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Thermo Scientific</td>
			<td>P4205987</td>
			<td></td>
		</tr>
		<tr>
			<td>C2C12 cells</td>
			<td>ATCC</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Chemical</td>
			<td>183172</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Bioreagents</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (Amplification Grade)</td>
			<td>Invitrogen</td>
			<td>18068015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>EMD Millipore</td>
			<td>MAB374</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>VWR Life Sciences</td>
			<td>19C2656013</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti-mouse secondary antibody (IRDYE 800CW)</td>
			<td>Li-Cor</td>
			<td>C90130-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti mouse secondary antibody (Rhodamine-red)</td>
			<td>Jackson Immune Research</td>
			<td>133389</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Chemical</td>
			<td>A144S</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (Shaker)</td>
			<td>Denville Scientific Corporation</td>
			<td>1704N205BC105</td>
			<td></td>
		</tr>
		<tr>
			<td>Mercaptoethanol</td>
			<td>Amresco, VWR Life Sciences</td>
			<td>2707C122</td>
			<td></td>
		</tr>
		<tr>
			<td>Midiprep plasmid extraction kit</td>
			<td>Qiagen</td>
			<td>12643</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin 4 (myosin heavy chain)</td>
			<td>Invitrogen</td>
			<td>14-6503-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Invitrogen</td>
			<td>2086310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR Life Sciences</td>
			<td>241</td>
			<td></td>
		</tr>
		<tr>
			<td>non-ionic surfactant/detergent</td>
			<td>VWR Life Sciences</td>
			<td>18D1856500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MP</td>
			<td>199983</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Bioreagents</td>
			<td>BP399-4</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin antibiotics</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petridishes</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>Fisherbrand</td>
			<td>149569C</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>33576300</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Fisher Scientific</td>
			<td>BP2956100</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR (Real Time)</td>
			<td>Applied Biosystems</td>
			<td>4359284</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcription Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>Thermo Scientific</td>
			<td>TK274910</td>
			<td></td>
		</tr>
		<tr>
			<td>sh control plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>07201820MN</td>
			<td></td>
		</tr>
		<tr>
			<td>sh 3086 plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>TRCN0000092578</td>
			<td></td>
		</tr>
		<tr>
			<td>sh 972 plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>TRCN0000092579</td>
			<td></td>
		</tr>
		<tr>
			<td>sh 1977 plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>TRCN0000092582</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer (Nanodrop)</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop One C</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green Reagent Master Mix</td>
			<td>Applied Biosystems</td>
			<td>743566</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Acros Organics</td>
			<td>30145369</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol reagent</td>
			<td>Ambion</td>
			<td>254707</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>GIBCO</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher Bioreagents</td>
			<td>BP1421</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.25%</td>
			<td>Gibco-Life Technology Corporation</td>
			<td>2085459</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (DEPC treated and nuclease free)</td>
			<td>Fisher Bioreagents</td>
			<td>186163</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blotting apparatus</td>
			<td>Biorad</td>
			<td>Mini Protean Tetra Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Bioreagents</td>
			<td>BP1422</td>
			<td></td>
		</tr>
	</tbody>
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stable knockdown of genes encoding extracellular matrix proteins in the c2c12 myoblast cell line using small-hairpin (sh)rna

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Selection of puromycin-resistant C2C12 can be achieved in 10&#8722;14 days after transfection due to efficient elimination of non-resistant, i.e., untransfected cells (Figure 1B). Typically, more than 80% of the cells detach from the cell culture dish and these cells are removed during routine cell maintenance. Puromycin-resistant C2C12 cells expressing the control (scrambled) shRNA retain the spindle-shape, elongated cell morphology at low cell density and the capability to...

Watch this Scientific Journal Video about Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA at JoVE.com

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

This protocol is significant because it allows us to study the function of proteins, such as extracellular matrix proteins, that are up-regulated in later stages of myotube formation or maturation. This method can be used to knock down any gene of interest in C2C12 cells by using specific shRNAs and the respective analytical tools for phenotyping. The main advantage of this method is that we have generated C2C12 cells that can continuously suppress our genes of interest, even in mature myotubes.The most important aspect of this procedure is to maintain C2C12 cells in the undifferentiated state, which means at a low cell density during routine cell culture. The day before transfection, seed five times 10 to the fourth C2C12 cells per milliliter in two milliliters of complete DMEM per well in a six-well plate to achieve a 40 to 50%confluence after overnight incubation at 37 degrees Celsius and 5%CO2. The next morning, combine 100 microliters of 25-millimolar sodium chloride in one 1.5-milliliter reaction tube per culture well with 25.5 microliters PEI stock solution of an undifferentiated C2C12 cell culture for a five-minute incubation at 37 degrees Celsius.Next, combine three micrograms of plasmid DNA with 100 microliters of 25-millimolar sodium chloride per culture well for a five-minute incubation at 37 degrees Celsius. At the end of the incubations, combine the entire volume of each diluted PEI reagent with each diluted plasmid solution with gentle pipetting for a 25-minute incubation at 37 degrees Celsius. During the incubation, replace the supernatants of the C2C12 cell cultures with DMEM without antibiotics or serum.At the end of the incubation, add the entire volume of each transfection mix to each well in droplets, while continuously moving the cell culture dish. After six hours in the cell culture incubator, replace the medium in each well with complete DMEM. 24 hours after the transfection, replace the complete DMEM in each well with selection medium supplemented with five micrograms per milliliter of puromycin, and return the cells to the cell culture incubator for 10 to 14 days or until stable C2C12 cells are observed.Then expand the puromycin-resistant C2C12 cells in low cell density cultures in the presence of five micrograms per milliliter of puromycin, and cryopreserve six to 10 vials of cells for future use. To prepare the C2C12 cells for differentiation, seed 1.5 times 10 to the fifth stable puromycin-resistant C2C12 cells in one milliliter of selection medium per well in a 12-well plate, and incubate at 37 degrees Celsius and 5%carbon dioxide until the cultures are 95%confluent. To induce differentiation, replace the medium in each well with serum-free DMEM every two days following the myotube formation on an inverted bright-field microscope equipped with a camera.For myosin heavy chain immunostaining of the differentiated cells, seed five times 10 to the fourth puromycin-resistant C2C12 cells in 500 microliters of complete DMEM per chamber onto an eight-well chamber slide, and return the cells to the cell culture incubator. When the cells reach 95%confluency, replace the selection medium in each well with 500 microliters of serum-free DMEM. At the appropriate experimental time points, rinse the differentiating cells in each well three times with 5 milliliters of PBS per wash before fixing the cells with 200 microliters of 4%paraformaldehyde in PBS per well.After 15 minutes, wash the cells three times with fresh PBS per wash as just demonstrated followed by incubation with 200 microliters of 5-molar glycine in PBS per well for five minutes. At the end of the incubation, wash the cells three times before permeabilizing the cells with 200 microliters of 1%non-ionic surfactant in PBS for 10 minutes. Next, block the nonspecific antibody binding sites with 200 microliters of 5%bovine serum albumin in PBS per well for one hour followed by three washes in PBS.After the last wash, label the cells with 200 microliters of myosin heavy chain antibody for two hours at room temperature. After three PBS washes, incubate the cells with the appropriate secondary antibody for two hours at room temperature, protected from light. After three final washes, aspirate the PBS, and mount the cells with one drop of mounting medium supplemented with DAPI and a coverslip.Then observe each chamber on a fluorescence microscope using the appropriate filter set. To assess the knockdown efficiency in the cell cultures by western blotting, first seed three times 10 to the fifth puromycin-resistant C2C12 cells in two milliliters of complete DMEM per well in a six-well plate. After 24 hours, rinse the cultures with PBS, and initiate differentiation of the cells with two milliliters of serum-free DMEM as demonstrated.For analysis of the secreted proteins at the appropriate experimental time points, collect one milliliter of serum-free conditioned medium from each well into individual 1.5-milliliter reaction tubes for centrifugation. After centrifugation, transfer one milliliter of the conditioned medium supernatants into new 1.5-milliliter reaction tubes, and precipitate the proteins with 391 microliters of trichloroacetic acid in non-ionic surfactant per tube. After 30 seconds of vortexing, incubate the mixtures on ice for 10 minutes.At the end of the incubation, pellet the precipitated proteins by centrifugation, and wash the protein pellets three times with fresh ice-cold acetone and one centrifugation per wash. After the last wash, air-dry the pellets for three to four minutes at room temperature before resuspension in 50 microliters of SDS-PAGE sample buffer per tube. Then boil the samples for five minutes at 95 degrees Celsius before western blot analysis according to standard protocols.For intracellular and membrane-bound protein analysis, rinse the cell layer in each well with two milliliters of PBS per well, and use a cell scraper to carefully dislodge the cells in one-milliliter volumes of PBS. Transfer the cells into individual 1.5-milliliter tubes for centrifugation followed by three washes in one milliliter of PBS per tube per wash via centrifugation. After the last wash, resuspend the cell pellets in 200 microliters of lysis buffer supplemented with EDTA-free protease inhibitor cocktail reagent for a 30-minute incubation on ice.At the end of the incubation, ultrasonicate the samples for 15 seconds on ice with a power output setting of 10 at an operating frequency of 23 kilohertz, and remove the cell debris by centrifugation. Transfer the supernatants into new 1.5-milliliter reaction tubes, and determine the protein concentrations according to standard protocols. Combine 100 micrograms of protein with 5X SDS-PAGE sample buffer in a total volume of 60 microliters per sample, and boil the samples for five minutes at 95 degrees Celsius.Then analyze the samples by western blotting for the presence of the desired target protein. The selection of puromycin-resistant C2C12 cells can be achieved within 10 to 14 days of transfection due to an efficient elimination of the non-resistant cells. Typically, more than 80%of the C2C12 cells detach from the cell culture dish during this time period and are removed during routine cell maintenance.Puromycin-resistant C2C12 cells expressing the control or the ADAMTSL2 shRNA retain their spindle-shaped, elongated cell morphology at a low cell density, as well as their ability to differentiate into myotubes. C2C12 differentiation upon serum withdrawal can be monitored by bright-field microscopy and immunostaining for the myotube marker myosin heavy chain. Myosin heavy chain-positive myotubes are observed between three to five days after differentiation initiation and are multinucleated, as observed by the presence of more than one DAPI-positive nucleus within the cell boundaries.As the gene expression data in proliferation conditions demonstrate, the knockdown efficiency of the transfection is 40 to 60%Western blot analysis can be performed to confirm the success of the knockdown in cell lysates obtained, for example, from C2C12 cells stably expressing shRNA that targets ADAMTSL2 compared to control shRNA expressing cells. Be sure to maintain the puromycin concentration during the selection process high enough to eliminate all of the non-transfected C2C12 cells, or the non-transfected cells may outgrow the transfected cells. This assay allows us to determine the function of extracellular matrix proteins that are expressed later in muscle development, even if they are induced five or 10 days after C2C12 differentiation.Remember that the RNA extraction reagent and beta-mercaptoethanol should be handled in a fume hood and to handle the trichloroacetic acid according to the appropriate safety protocols. Thanks for watching, and good luck with your experiment.

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