We are grateful to Drs. Mike S. Brown and Joseph L. Goldstein for their agreement to publish this method according to the methods described in their publications. We appreciate Dr. Peter Espenshade for sharing the GFP-SCAP plasmid. This work was supported by NIH grants NS072838 and NS079701 to D.G., American Cancer Society Research Scholar Grant RSG-14-228-01-CSM to D.G., and OSUCCC Pelotonia Postdoctoral Fellowship to C.C. We also appreciate the support from the Ohio State Neuroscience Core (P30 NS038526) and OSUCCC Translational Therapeutic Program seed grant and start-up funds to D.G.

İnsan hücrelerinde 1. Tespit endojen SCAP Protein <ol><li> Hücre Kültürü ve İşlem <ol><li> Tohum yaklaşık 1 x 10 6 U87% 5 fetal sığır serumu (FBS) ile desteklenmiş Dulbecco Modifiye Edilmiş Eagle Ortamı (DMEM) ile 10 cm&#39;lik bir tabak hücreleri ve tedaviden önce 24 saat boyunca CO2, 37 ° C&#39;de inkübe hücreleri ve% 5. </li><li> fosfat tamponlu tuzlu su (PBS) ile bir kez hücreler yıkanır, daha sonra 12 saat boyunca (5 mM) ile birlikte ya da glükoz ihtiva etmeyen taze DMEM ortamı hücreleri geçer. </li></ol></li><li> Hücre membran fraksiyonları ve Nükleer özütlerin hazırlanması <ol><li> Yıkama hücreler bir kez PBS ile 4 ° C &#39;de 5 dakika boyunca 1000 x g&#39;de 1 mi PBS içine kazıma ve santrifüjlenir. </li><li> 10 mM HEPES-KOH (pH 7.6), 10 mM KCI, 1.5 mM MgCI2, 1 mM sodyum etilendiamintetraasetik asit (EDTA), 1 mM sodyum etilen glikol tetraasetik asit (EGTA), 250 mM ihtiva eden buzla soğutulmuş bir tampon maddesi içinde yeniden süspanse hücreleri sakroz ve proteaz inhibitörleri karışımı (5 μug / ml pepstatin A, 10 ug / ml löpeptin, 0.5 mM fenilmetilsülfonil florid (PMSF), 1 mM ditiotreitol (DTT) ve 25 ug / ml N-asetil-Leu-Leu-norLeu-al, 30 dakika boyunca (ALLN)) buzda. </li><li> çekirdekleri izole etmek için 5 dakika boyunca 4 ° C&#39;de 890 x g&#39;de 22 x g ile 1 1/2 iğne 30 kez ve santrifüj hücre ekstreleri geçirin. </li><li> Membran fraksiyonlarının (aşama 1.2.7) ayrılması için supernatant kullanın. Tampon C (20 mM HEPES / KOH, pH 7.6, 0.42 M NaCl,% 2.5 (h / h) gliserol, 0.1 mi nükleer pelet yeniden süspanse edin, 1.5 mM MgCI2, 1 mM sodyum EDTA, 1 mM sodyum EGTA ve karışım proteaz inhibitörleri (5 ug / ml pepstatin A, 10 ug / ml löpeptin, 0.5 mM PMSF, 1 mM DTT ve 25 ug / ml ALLN)). </li><li> 4 ° C&#39;de 20 dakika boyunca bir mikrosantrifüj içinde 20,000 x g&#39;de 60 dakika santrifüj boyunca 4 ° C &#39;de süspansiyon döndürün. süpernatant &quot;nükleer özler&quot; olarak belirlenmiştir. </li><li> (5x yükleme tamponu ile 10 dakika boyunca 31 100 ° C&#39;de çekirdek özütleri IsıSDS-PAGE maruz kalmadan önce 2.5 mM Tris-HCI, pH 6.8,% 10 SDS,% 50 gliserol,% 12.5 β-mersaptoetanol ve mavi,% 0.05 bromofenol). </li><li> 4 ° C&#39;de 20 dakika boyunca 20,000 x g&#39;de orijinal 890 xg dönüş süpernatant santrifüjleyin. daha sonra Western blot analizi için, 0.1 SDS liziz tamponu (10 mM Tris-HCI pH 6.8, 100 mM NaCI,% 1 (h / h) sodyum dodesil sülfat (SDS), 1 mM sodyum EDTA ve 1 pelet çözülür mM sodyum EGTA). Bu, &quot;membran fraksiyonu&quot; olarak adlandırılır. NOT: membranlar pelet 20.000 xg 20 dakika kullanın. 10 100,000 xg - 20 dakika Brown ve Goldstein, laboratuarda 15 ile kağıt kullanılmaktadır. </li><li> 30 dakika boyunca 37 ° C &#39;de membran fraksiyonu inkübe ve Bradford protein tahliliyle protein konsantrasyonu belirlenir. SDS-PAGE için numuneler tabi önce 1 ul 100x bromofenol mavi çözeltisi ekleyin. </li><li> % 10 SDS-PAGE jeli 39 toplam membran proteinlerinin 50 ug - Yük 25. jel çalıştırın80 V 15 dakika, daha sonra da 130 V değiştirmek ve başka bir 115 dakika boyunca çalışır. 130 dakika 39 için 140 mA aktarın. <ol><li> Western blot analizi için, toplam SCAP proteini tespit etmek için bir anti-SCAP antikoru kullanımı. Bir iç kontrol olarak, protein disülfid izomeraz (KAE), bir ER-yerleşik protein 40 kullanın. SREBP-1 N-terminal grubu tespit etmek için bir anti-SREBP-1 antikoru kullanın. Nükleer ekstreler 31 için bir iç kontrol olarak lamin A kullanın. </li></ol></li></ol></li></ol> İnsan hücrelerinde 2. Analiz Scap N -glycosylation <ol><li> Hücre Kültürü, transfeksiyon, ve Tedavi <ol><li> Membran analizi için, DMEM ile 10 cm&#39;lik bir tabak tohum yaklaşık 1 x 10 6 HEK293T hücreleri,% 5 FBS ile takviye edilmiş ve transfeksiyondan 24 saat önce CO2, 37 ° C&#39;de inkübe hücreleri ve% 5. </li><li> Serum orta azaltılmış 1 ml 8 mg GFP-SCAP plazmid (Dr. Peter Espenshade bir hediye) 22 ve hafifçe karıştırın - 4 seyreltin. </li><li> plazmaları ihtiva serum orta azaltılmış 1 ml doğrudan pipetleme 16 ul DNA transfeksiyon ayıracı ve hafifçe karıştırın - 8 ekleyin. </li><li> Oda sıcaklığında 25 dakika boyunca transfeksiyon reaktifi / plazmid kompleksi inkübe edin. </li><li> Taze ortam ile hücrelere transfeksiyon kompleksi ilave edin ve% 5 CO2 ve 37 ° C&#39;de 24 saat boyunca kültüre devam etmektedir. </li><li> Bir kez PBS ile yıkayın ve veya tunikamisin (1 ug / ml), bir N -glycosylation inhibitörün varlığında veya yokluğunda, glikoz (5 mM) olmaksızın 12 saat boyunca hücre tedavisi. </li></ol></li><li> adımlar 1.2.1, 1.2.2, 1.2.3 ve 1.2.7 de açıklandığı gibi hücre zarı pelet hazırlayın. </li><li> SCAP N -glycosylation tespiti <ol><li> tripsin Proteoliz <ol><li> 10 mM HEPES · KOH (pH 7.4), 10 mM KCI, 1.5 mM MgCI2, 1 mM sodyum EDTA ve 100 mM NaCl ihtiva eden tampon 114 ul GFP SCAP aşırı ifade eden hücrelerden membran pelet yeniden süspanse edin. </li><li> 57 inkübe81; 30 ° C &#39;de 30 dakika boyunca 58 ul&#39;lik bir toplam hacimde, yokluğu veya tripsin 1 ul (1 ug / ml) varlığında membran proteinlerinin l tümbölenler. </li><li> soya fasulyesi tripsin inhibitörü 2 ul (400 birim) eklenerek reaksiyonlar durur. </li></ol></li><li> Peptid N glikosidaz F deglikosilasyon (PNGase F) <ol><li> PNGase F ile muamele için,% 3.5 (ağırlık / hacim) SDS ve% 7 (hacim / hacim) ihtiva eden bir çözeltiden 10 ul her bir örnek için 2-merkaptoetanol ekleyin. </li><li> 10 dakika boyunca 100 ° C&#39;de ısıtıldıktan sonra, sırayla, 0.5 M sodyum fosfat (pH 7.5),% 10 (h / h) Nonidet 12 x proteaz inhibitörleri P-40 ve 2 ul 7 ul 7 ul (0.5 U ekleme PNGase F. / ml) </li><li> 3 saat 37 ° C&#39;de inkübasyondan sonra, 5x yükleme tamponu (312.5 mM Tris-HCI pH 6.8,% 10 SDS,% 50 gliserol,% 12.5 β-merkaptoetanol ve% 0.05 bromofenol mavisi) eklenerek reaksiyonlar durur. 10 m, 100 ° C&#39;de karışımın ısı İleve SDS-PAGE tabi. % 10 SDS-PAGE jeli 39 toplam membran proteinlerinin yük 25-50 ug. daha sonra başka bir 115 dakika boyunca 130 V değiştirmek 15 dakika boyunca 80 V jel üzerinde çalıştırıldı. 130 dakika boyunca 140 mA aktarın. </li><li> Western Blot için, N -glycosylation toplam SCAP proteini tespit etmek için 10 ug / ml anti-SCAP (9D5) antikoru kullanımı. </li></ol></li></ol></li></ol> İnsan hücrelerinde GFP-SCAP kullanarak Algılama Golgi ER SCAP Ticareti 3. <ol><li> DMEM ile 60 mm çanak Tohum 2.5 x 10 5 U87 hücreler% 5 FBS ile takviye edilmiş ve transfeksiyondan 24 saat önce CO2, 37 ° C&#39;de inkübe hücreleri ve% 5. </li><li> 0.5 ml 4 mg GFP-SCAP plazmid azaltılmış serum orta sulandırmak ve karıştırın. </li><li> şirketinden 0.5 ml plazmidleri içeren düşük serum ortamı pipetleme 8 ul DNA transfeksiyon reaktifi ekleyin ve yavaşça karıştırın. </li><li> oda 25 dakika boyunca transfeksiyon reaktifi / plazmid kompleksi inkübesıcaklık. </li><li> Taze ortam ile hücrelere transfeksiyon kompleksi ilave edin ve% 5 CO2 ve 37 ° C&#39;de 24 saat boyunca kültüre devam etmektedir. </li><li> Cam kapak kayma (22 mm x 22 mm) ihtiva eden bir 6-yuvalı plaka 10 x 10 4 hücreleri ve 24 saat boyunca 37 ° C ve% 5 CO2 kültüre devam - 5 reseed. </li><li> Bir kez PBS ile yıkayın ve veya tunikamisin (1 ug / ml) varlığında ya da yokluğunda, glikoz (5 mM) olmaksızın 12 saat boyunca hücre tedavisi. </li><li> Bir kez PBS ile yıkayın hücreleri ve 10 dakika boyunca% 4 formaldehit içinde çözmek. </li><li> , Lamelleri ters, PBS ile hücreleri üç kez yıkayın antifade reaktif ile birlikte slaytlar monte ve tırnak cilası kullanarak lamelleri mühür. </li><li> Bir lazer tarama konfokal mikroskop 41 63X immersiyon yağı hedefi kullanarak GFP-SCAP ticaretini kontrol edin. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocatalytic+processing+of+site-1+protease+removes+propeptide+and+permits+cleavage+of+sterol+regulatory+element-binding+proteins.">Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.</a> The Journal of biological chemistry. 274, 22795-22804 (1999).</li><li>Guo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCAP+links+glucose+to+lipid+metabolism+in+cancer+cells.">SCAP links glucose to lipid metabolism in cancer cells.</a> Mol Cell Oncol. 3, (2016).</li><li>Cheng, 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=Glucose-Mediated+N-glycosylation+of+SCAP+Is+Essential+for+SREBP-1+Activation+and+Tumor+Growth.">Glucose-Mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth.</a> Cancer Cell. 28, 569-581 (2015).</li><li>Shao, W., Espenshade, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+Makes+Fat+by+Talking+to+SCAP.">Sugar Makes Fat by Talking to SCAP.</a> Cancer Cell. 28, 548-549 (2015).</li><li>Guo, 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=EGFR+signaling+through+an+Akt-SREBP-1-dependent,+rapamycin-resistant+pathway+sensitizes+glioblastomas+to+antilipogenic+therapy.">EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.</a> Sci Signal. 2, 82 (2009).</li><li>Guo, 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=An+LXR+agonist+promotes+GBM+cell+death+through+inhibition+of+an+EGFR/AKT/SREBP-1/LDLR-dependent+pathway.">An LXR agonist promotes GBM cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway.</a> Cancer Discov. 1, 442-456 (2011).</li><li>Guo, D., Bell, E. H., Mischel, P., Chakravarti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+SREBP-1-driven+lipid+metabolism+to+treat+cancer.">Targeting SREBP-1-driven lipid metabolism to treat cancer.</a> Curr Pharm Des. 20, 2619-2626 (2014).</li><li>Bell, E. H., Guo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+for+malignant+gliomas.">Biomarkers for malignant gliomas.</a> Malignant Gliomas, Radiation Medicine Rounds. 3, 339-357 (2012).</li><li>Geng, F., 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=Inhibition+of+SOAT1+suppresses+glioblastoma+growth+via+blocking+SREBP-1-mediated+lipogenesis.">Inhibition of SOAT1 suppresses glioblastoma growth via blocking SREBP-1-mediated lipogenesis.</a> Clin Cancer Res. , (2016).</li><li>Ru, 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=Feedback+Loop+Regulation+of+SCAP/SREBP-1+by+miR-29+Modulates+EGFR+Signaling-Driven+Glioblastoma+Growth.">Feedback Loop Regulation of SCAP/SREBP-1 by miR-29 Modulates EGFR Signaling-Driven Glioblastoma Growth.</a> Cell Rep. , (2016).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual (Fourth Edition). , (2012).</li><li>Uehara, T., 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=S-nitrosylated+protein-disulphide+isomerase+links+protein+misfolding+to+neurodegeneration.">S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration.</a> Nature. 441, 513-517 (2006).</li><li>Miyashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy+for+intracellular+co-localization+of+proteins.">Confocal microscopy for intracellular co-localization of proteins.</a> Methods Mol Biol. 1278, 515-526 (2015).</li><li>Cao, J., Guo, S., Arai, K., Lo, E. H., Ning, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+extracellular+signaling+utilizing+a+glycoproteomic+approach:+lectin+blot+surveys,+a+first+and+important+step.">Studying extracellular signaling utilizing a glycoproteomic approach: lectin blot surveys, a first and important step.</a> Methods Mol Biol. 1013, 227-233 (2013).</li></ol>

The authors have nothing to disclose.

Bu çalışmada, insan hücrelerinde membran fraksiyonlarının izolasyonu için ve Western blot ile SCAP N -glycosylation ve toplam proteinin belirlenmesi için numune hazırlanması için bir protokol açıklar. Biz daha GFP-etiketleme ve konfokal mikroskopi kullanılarak SCAP kaçakçılığı izlemek için bir yöntem sağlar. Yöntem özellikle membran proteini analiz etmek için kullanılan ve SCAP N -glycosylation ve insan ticaretini soruşturma önemli bir araçtır. Farklı N -glycan zincirleri tanır ve N- -glycosylated proteinleri 42 tespit için çeşitli lektinler kullanılarak yöntemi ile karşılaştırıldığında, burada tarif edilen eden bir yöntem SCAP protein parçacığı glikosilasyonu belirlemek için geçiş Shift proteine bağlı N -glycan kaldırmak için PNGase F kullanır tespit aa 540-707. lektin yönteminin sınırlama belirli tanımak için lektin uygun tip belirlenmesi bağlıdır <em&gt; K -glycan. Bizim PNGase F bölünme yöntemi SCAP N -glycosylation analiz etmek için basit bir prosedür sağlar. Bundan başka, yöntem, aynı zamanda homojenleştirme sonrası 2, insan hastalar veya hayvan dokularında SCAP N -glycosylation ve protein seviyesini analiz etmek için uygulanabilir. Çalışmamız kanser ve metabolik hastalıklarda lipid metabolizması imza analiz etmek için yeni bir araç sağlar. Bu çalışmada, insan hücrelerinde N -glycosylation ve trafik analizinde kullanılan GFP etiketli Scap plasmid hamster kökenlidir. SCAP proteininin 540-707 fragmanı, sadece protein ve Çin hamsteri yumurtalık (CHO) hücre hattı 15 deki gibi bir hamster Scap N -glycosylation algılayabilir amino asitlerine karşı yükseltilmiş SCAP antikoru (IgG-9D5). Amino asitlerine karşı antikor 450 - Şekil 1 &#39;de gösterildiği gibi, insan Scap 500, insan hücrelerinde endojen SCAP proteinini tespit edebilmektedir SCAP N -glyco tespit etmek.İnsan Scap 707 fragmanı - insan hücreleri veya dokularda sylation, amino asitlerin karşı 540, belirli bir antikor geliştirmek gerekir. Buna ek olarak, belirli bir lektin tanımlanması SCAP-bağlı N -glycan insan SCAP N -glycosylation 42 analiz için alternatif bir yöntemdir tanımak için. Ayrıca, SCAP bağlama her N -glycan zincirin bileşimi ve yapısını tanımlayan (N263, N590 ve N641) Golgi ER SCAP ticareti yatan mekanizma anlayışımızı kolaylaştıracaktır. Ayrıca, SCAP N -glycosylation saptanması için en iyi sonuçları elde etmek için, Brown ve Goldstein Laboratory 15 ile yayınlarda tarif edilen yöntemlerle değiştirilmiş olan deneysel koşullara göre bir tepki parametreleri ayarlanabilir. Başarılı bir şekilde SCAP protein ve bunun N -glycosylation tespit etmek için, membran fraksiyonları ve nükleer özler kalitesi çok önemlidir. Bu, doğrudan buz üzerine örnek koyarak kaçınarak 37 ° C&#39;de inkübe membran fraksiyonları sonra protein konsantrasyonu belirlenir. Burada tarif edilen yöntem yaygın kanser, kalp-damar hastalığı, diyabet ve obezite de dahil olmak üzere farklı araştırma alanları uygulanabilir.

Lipid metabolizması deregülasyon kanseri ve metabolizma hastalıkları 1-6 ortak bir özelliğidir. Bu işlemlerde, sterol düzenleyici elementi bağlayan proteinler (SREBPs), transkripsiyon faktörleri ailesidir, alım ve kolesterol, yağlı asitlerin sentezi ve fosfolipid 7-10 önemli genlerin ekspresyonunu kontrol etmede çok önemli bir rol oynar. SREBP-1 a, SREBP-1 c ve SREBP-2 de dahil olmak üzere SREBPs, iki transmembran 7 sayesinde endoplazmik retikulum (ER) zarına bağlanan etkin olmayan öncüler olarak sentezlenir. SREBPs N-terminali, hedef genlerinin 11 transaktivasyonu için bir DNA-bağlama alanını içerir. SREBP öncülerinin C-terminali SREBP bölünmesi aktive edici protein (SCAP) 12,13, SREBP stabilitesi ve aktivasyon 14-16 düzenlenmesinde çok önemli bir rol oynayan bir politopik membran proteinine bağlanır. implementranskripsiyonel fonksiyonunun yon iki proteazlar ardışık SREBP ayrılması ve daha sonra lipojenik genlerin 7 transaktivasyonu için çekirdeğin içine girer N-terminal fragmanı serbest Golgi, ER SCAP / SREBP kompleksinin, gerektirir. Bu işlemlerde, ER membran sterol seviyeleri ER 7,17 den SCAP / SREBP kompleksinin çıkış kontrol eder. Yüksek sterol koşullar altında, sterol Scap veya ER yerleşik bağlanan insülin ile uyarılan gen proteini-1 (INSIG-1) veya 2 (INSIG-2), içinde SCAP / SREBP kompleksi korumak Insigs ile Scap ilişkisini arttırıcı ER 18-20. sterol düzeyleri azalma olduğunda, SCAP Insigs ile ayrışmaktadır. Bu ortak kaplama proteinleri (COP) II kompleksi ile SCAP etkileşim sağlayan bir SCAP konformasyonel değişime yol açar. Karmaşık tomurcuklanma vezikülleri içine SCAP / SREBP kompleksi dahil edilmesine aracılık eder ve Golgi 21,22 ER; nakliye yönlendirir. TRANSLOC üzerineGolgi ation, SREBPs ardışık N-terminali 7,23-29 serbest kalmasına yol, alana, 1 ve-2 proteaz tarafından ayrılmaktadır. SCAP proteini N asparajinde oligosakkaritler -bağlı üç taşır (N) N263, N590, N641 ve 15 konumlandırır. Biz son zamanlarda bu sitelerde SCAP glukoz aracılı N -glycosylation düşük sterol koşullarında 30-32 altında Golgi ER SCAP / SREBP kaçakçılığı için bir ön koşul olduğunu ortaya koymuştur. (NNN qqq için) glutamin için üç asparagin mutasyon yoluyla SCAP glikosilasyon kaybı SCAP protein ve SREBP aktivasyon 31 azaltılması istikrarsızlık SCAP / SREBP kompleksinin ve sonuçların ticaretini devre dışı bırakır. Bizim son veriler de bu SREBP-1 yüksek glioblastoma aktive ve SCAP N -glycosylation 4,31,33,34 tarafından düzenlenir göstermektedir. Hedefleme SCAP / SREBP-1 sinyal maligniteler ve metabolik s tedavisinde yeni bir strateji olarak ortaya çıkmaktadır1,3,35-38 yndromes. Bu nedenle, SCAP proteini ve N -glycosylation düzeylerini analiz etmek ve insan hücreleri ve hasta dokularda kaçakçılığı izlemek için etkin bir yöntem geliştirmektir önemlidir. ER &#39;in içinde SCAP proteini (amino asitler 540-707) luminal bölgesi sağlam zarları tripsin 15 ile muamele edildiğinde proteoliz korunur iki N -glycosylation sitesi (N590 ve N641) içerir. Bu lümen parçası, sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) 15,31 ile Scap bireysel glikosile varyantlarının çözünürlüğünü sağlamak için yeterince küçüktür ~ 30 kDa&#39;lık bir moleküler ağırlığa sahiptir. Burada, N Scap arasında -glycosylation ve insan toplam hücre proteini tespit etmek için bir yöntem sağlar. Bu protokol, Brown ve Goldstein, laboratuarda 15 ve son yayın 31 &#39;den yayınlarda açıklanan yönteme elde edilir. Protokol t kullanılabilirO memeli hücrelerinden SCAP protein çalışma.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<colgroup>
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">X-treme GENE HP DNA Transfection Reagent&#160;</td>
			<td style="width:163px;">Roche</td>
			<td style="width:123px;">6366236001</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEMI medium&#160;</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypsin&#160;</td>
			<td>Sigma</td>
			<td>T6567</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PNGase F&#160;</td>
			<td>Sigma</td>
			<td>P7367</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SCAP (9D5) antibody&#160;&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-13553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP antibody&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SREBP-1 antibody (IgG-2A4)</td>
			<td>BD Pharmingen</td>
			<td>557036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDI Antibody (H-17)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-30932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lamin A Antibody (H-102)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-20680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCAP antibody (a.a 450-500)</td>
			<td>Bethyl Laboratories, Inc.</td>
			<td>A303-554A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) without glucose, pyruvate and glutamine&#160;&#160;</td>
			<td>Cellgro</td>
			<td>17-207-CV</td>
			<td>Add 1 Mm Pyravate and 4 mM Glutamine before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G x 1 1/2 needle&#160;</td>
			<td>BD&#160;</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pepstatin A</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ALLN</td>
			<td>Sigma</td>
			<td>A6185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>GE</td>
			<td>RPN3032D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA Solution 0.5 M PH8.5 100 ml&#160;</td>
			<td>VWR</td>
			<td>82023-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA 0.5 M sterile (PH 8.0) 50 ml</td>
			<td>Fisher Scentific</td>
			<td>50255956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone FBS</td>
			<td>Thermo scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bromophenol blue</td>
			<td>Sigma</td>
			<td>B8026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">prolong gold antifade reagent with dapi</td>
			<td>life technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td>life technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td>life technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of scap n-glycosylation and trafficking in human cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Şekil 1, Western blot Glükoz uyarısına karşı tepki olarak insan glioblastoma U87 hücrelerinde endojen SCAP protein ve SREBP-1 nükleer formunun belirlenmesini göstermektedir. zar proteini SCAP &quot;zar fraksiyonu&quot; tespit edilir. SREBP-1 çekirdeği bir şekilde (N-terminal) &quot;Nükleer özler&quot; tespit edilir. SCAP protein düzeyi belirgin glükoz uyarılması ile geliştirilmiş ve SREBP-1 nükleer formu (KAE bir ER membran proteinlerinin iç kontrol olarak hizmet vermektedir). Bu sonuçlar, bir protokol, insan hücrelerinde endojen SCAP proteinini tespit etmek mümkün olduğunu göstermektedir. Şekil 2, GFP-etiketli Hamster SCAP ifade HEK293T hücrelerinde uyarılması ve tunikamisin tedavi glikoza tepki olarak SCAP N -glycosylation toplam GFP SCAP protein düzeylerinin analizini göstermektedir. Şekil <strong&gt; 2A üç N -glycosylation siteleri taşıyan ER zarından SCAP proteini tematik yapısını göstermektedir. AA 540 ihtiva eden Scap fragmanı - ER 6.cısı lümen döngüsünde bulunan 707, trypsin&#39;e dirençli 15 ve sitelerinin N590 ve N641 N -glycosylation analizi için kullanılır. Tedavi, (aa 540 ihtiva eden - 707) daha yüksek bir ağırlık bantı tripsin sonra Şekil 2B&#39;de (üst panel), gösterildiği gibi, bir ya da iki N PNGase glikoz ve yokluğunda mevcudiyetinde oligosakaritler (N590 ve N641) -bağlı ihtiva SCAP proteini belirtir (IgG 9D5 antikoru ile tespit) ya da tunikamisin. Daha hızlı SDS-PAGE hareket göre - PNGase F varlığında, N-bağlı oligosakaritler fragmanını (707 aa 540) neden SCAP protein çıkarıldı. Sürekli tamamen tunikamisin N -glycosylation başlatma işlemini engelleme alt görünüşü ile gösterilen SCAP N -glycosylation kaldırıldıSCAP fragmanı (Şekil 2B, üst levha) ve bir grup. Ayrıca, (GFP karşı bir antikor ile tespit edilir), toplam GFP SCAP proteini glukozun yokluğunda ya tunikamisin tedavisi (Şekil 2B, alt panel) ile indirgendi. Bu sonuçlar, bir protokol SCAP N -glycosylation analizi için uygun olduğunu göstermektedir. Şekil 3, Glükoz uyarısına karşı tepki Golgi ER GFP-SCAP trafik U87 hücrelerinin tunikamisin işlenerek bastırılmıştır göstermektedir. Şekil 3A&#39;da gösterildiği gibi, konfokal mikroskopi resim PDI, bir ER yerleşik bir protein (kırmızı) ile eş-lokalizasyonu ile gösterildiği gibi GFP SCAP proteini, glukozun yokluğunda ER yer aldığını göstermektedir. Bunun aksine, glükoz mevcudiyetinde GFP-SCAP Golgi proteini markör Giantin (kırmızı) (Şekil 3B) ile eş-lokalizasyonu ile gösterilen, Golgi hareket eder. Ayrıca, tunikamisinTedavi Golgi (Şekil 3A ve B) ER GFP-SCAP glikoz kaynaklı ticaretini bastırılır. <img alt="Şekil 1" src="/files/ftp_upload/54709/54709fig1.jpg" /> Şekil 1. Tespit Endojen SCAP Protein ve SREBP-1 U87 hücrelerde nükleer formu. Zar 12 saat (5 mM) ile ya da bir glukoz içermeyen serumsuz ortam içinde kültürlenen insan primer glioblastoma U87 hücrelerinin nükleer ekstrelerin Batı benek analizi. İnsan SCAP aa 450 karşı anti-SCAP antikoru - 500, insan hücrelerinde endojen SCAP tespit etmek için kullanıldı. İnsan SREBP-1 407 fragmanı - SREBP-1 aktif formu (N-terminal) AA 301 karşı antikor kullanılarak &quot;çekirdek özleri&quot; tespit edilir. Bu rakam, 31 izni ile modifiye edilmiştir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54709/54709fig1large.jpg" target="_blank">Daha büyük bir versi görmek için buraya tıklayınız</a>bu rakamın üzerinde. <img alt="şekil 2" src="/files/ftp_upload/54709/54709fig2.jpg" /> Şekil 2. SCAP N -glycosylation ve GFP-SCAP Protein Düzeylerinin Western Blot Analizi. A) SCAP yapısı yayılan ER zarı çapraz gösteren şematik diyagramı. ER lümeninde bulunan ve N-bağlı oligosakaritler modifiye bunların hepsi Scap üç asparagin (N) kalıntıları vardır. SCAP ihtiva eden amino asitler 540-707 fragman tripsin sindirime dirençlidir. . Bu aa 540 karşı IgG-9D5 antikoru ile tespit edilebilir, iki N-bağlantılı oligosakarit modifikasyonu sties, N590 ve N641 içerir - western blot B Hamster Scap) üst SCAP N -glycosylation (Western blot analizi 707: ile tripsin tedavisi) veya GFP-SCAP protein düzeyleri (alt: HEK293T hücrelerinden trans hiçbir tripsin tedavisi)iently 24 saat boyunca, GFP-Scap ile transfekte edilmiş ve 12 saat boyunca, ya da tunikamisin (1 ug / ml), N -glycosylation inhibitörün varlığında veya yokluğunda, glikoz (5 mM) olmadan, serumsuz ortamda kültür. Blot sol tarafındaki numaraları (IgG 9D5 antikoru ile tespit üst) SCAP protein (N590 ve N641 siteleri) N -glycosylation sitelerinin sayısını gösterir. GFP-SCAP için alt paneli GFP proteine ​​karşı bir antikor ile tespit edilmiştir. Bu rakam, 31 izni ile modifiye edilmiştir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54709/54709fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/54709/54709fig3.jpg" /> Golgi. A ER SCAP Ticareti Şekil 3. Algılama B) mikroskopisi görüntüleri GFP-SCAP ER bulunur veya t hareket ettiğini göstermektedirO 12 saat U87 hücrelerinin tunikamisin (1 ug / ml) tedavi olmadan / yokluğunda ya da glikoz (5 mM) varlığında, Golgi. PDI ER boyama (kırmızı) (A) göstermektedir. Giantin, Golgi markör (kırmızı) (B). DAPI (mavi) hücre çekirdeğine boyar. Ölçek çubuklar 10 mikron temsil etmektedir. Şekil 3A yeni veri ve daha önce yayınlanmamıştır. Şekil 3B 31 izni ile modifiye edilmiştir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54709/54709fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Bu Western blot ile SCAP N -glycosylation ve toplam proteinin belirlenmesi için insan hücrelerinden membran fraksiyonu izolasyonu ve numune hazırlama yönelik değişik bir metot tarif etmektedir. Biz daha konfokal mikroskopi kullanılarak SCAP ticaretini izlemek için bir GFP-etiketleme yöntemi tanıtmak. Bu protokol, normal bir biyolojik laboratuvarlarda kullanılabilir.

Scap analizi<em&gt; K</em&gt; İnsan Hücreleri -glycosylation ve Ticareti

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

analysis-of-scap-n-glycosylation-and-trafficking-in-human-cells

Nanyang Technological University

Research

JoVE Journal

Biology

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

8.6K Görüntüleme.  The Ohio State University Comprehensive Cancer Center and College of Medicine.  Bu Western blot ile SCAP N -glycosylation ve toplam proteinin belirlenmesi için insan hücrelerinden membran fraksiyonu izolasyonu ve numune hazırlama yönelik değişik bir metot tarif etmektedir. Biz daha konfokal mikroskopi kullanılarak SCAP ticaretini izlemek için bir GFP-etiketleme yöntemi tanıtmak. Bu protokol, normal bir biyolojik laboratuvarlarda kullanılabilir. 

Video: Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Biyoloji

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Sendai-virüs ile İnsan Somatik Verimli Üretimi İnsan uyarılmış pluripotent kök hücrelerin

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Kantitatif RT-PCR ile Bireysel İnsan Embriyonik Kök Hücreleri profilleme

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Colocalizational Analizi Reseptör Sonrası içselleştirme Ticareti Uyuşturucu kaynaklı Değişiklikleri İzleme

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

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.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

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

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Konfokal Mikroskobu kullanılarak Transfekte İnsan Embriyonik Böbrek Hücreleri Canlı Hücre Görüntüleme ve 3D Analiz Angiotensin Reseptör Tip 1a Ticareti

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

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.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

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

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

Vivo Algılama ve Rb Protein SUMOylation insan hücrelerinde analizi

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...