Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells

Mariusz R. Wieckowski1, Cláudia M. Deus2, Renata Couto2, Monika Oparka1, Magdalena Lebiedzińska‐Arciszewska1, Jerzy Duszyński1, Paulo J. Oliveira2

1 Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, 2 CNC—Center for Neuroscience and Cell Biology, University of Coimbra, Cantanhede
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.6
DOI:  10.1002/0471140856.tx2506s66
Online Posting Date:  November, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased phosphorylation of serine 36 residue of p66Shc very often correlates with oxidative stress–associated pathologies. The pro‐oxidative role of p66Shc also appears to be involved in chemical toxicity, being an important component of stress responses triggered by xenobiotics. Here, we present a protocol that can be used: (a) for isolation of mitochondrial, cytosolic, and mitochondrial‐associated membrane fractions from adherent cells lines; (b) to perform p66Shc detection with specific antibodies in order to monitor its translocation between different cellular compartments in response to the oxidative stress; and (c) to modulate the p66Shc pathway with the use of pharmacological approaches or gene‐silencing methods. © 2015 by John Wiley & Sons, Inc.

Keywords: cell signaling pathways; mitochondria; oxidative stress; protein phosphorylation and p66Shc protein

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Mitochondrial and Cytosolic Fractions
  • Basic Protocol 2: Isolation of Mitochondrial‐Associated Membranes
  • Basic Protocol 3: Immunoblotting of p66Shc and pSer36‐p66Shc in Different Fractions
  • Alternate Protocol 1: Immunoblotting for Activity of p66Shc Pathway‐Associated Proteins
  • Support Protocol 1: Pharmacological Inhibition of p66Shc Pathway
  • Support Protocol 2: Knockdown of p66Shc in Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Mitochondrial and Cytosolic Fractions

  Materials
  • Cells of interest
  • Appropriate growth medium for cells, containing 10% fetal bovine serum (FBS)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 0.05% (w/v) trypsin‐EDTA (Invitrogen, cat. no. 25300‐062)
  • Homogenization medium (see recipe)
  • Mitochondria isolation medium (see recipe)
  • Mitochondria resuspension buffer (see recipe)
  • 10‐cm2 cell culture dishes
  • 15‐ml conical polypropylene tubes (e.g., Corning Falcon)
  • Refrigerated low‐speed tabletop centrifuge (e.g., Sigma Model 2K15)
  • Stirrer motor with electronic speed controller
  • Motor‐driven tightly fitting glass/Teflon Potter‐ Elvehjem homogenizer
  • 30‐ml polypropylene centrifugation tubes
  • Ultracentrifuge (e.g., Beckman Coulter Optima L‐100 XP)
  • Additional reagents and equipment for pharmacological inhibition ( protocol 5) or knockdown ( protocol 6) of p66She pathway

Basic Protocol 2: Isolation of Mitochondrial‐Associated Membranes

  Materials
  • Crude mitochondrial fraction ( protocol 1)
  • MAM isolation buffer I (see recipe)
  • Mitochondria resuspension buffer (see recipe)
  • Ultra‐Clear 14‐ml polyallomer ultracentrifuge tubes
  • Ultracentrifuge: e.g., Beckman Coulter Optima L‐100 XP with, e.g., SW 40 Ti rotor, swinging‐bucket, 6 × 14 ml, and Beckman Type 70 Ti rotor, fixed‐angle, 8 × 39 ml
  • Refrigerated low‐speed centrifuge
  • 30‐ml tubes for refrigerated low‐speed centrifuge
  • Polycarbonate tubes with cap assembly (Beckman, cat. no. 355618, for use with 70Ti rotor)

Basic Protocol 3: Immunoblotting of p66Shc and pSer36‐p66Shc in Different Fractions

  Materials
  • Mitochondrial, cytosolic, and MAM fractions (see Basic Protocols protocol 11 and protocol 22, respectively)
  • 10× Cell Lysis Buffer (Cell Signaling, cat. no. 1679803S)
  • Phenylmethylsulfonyl fluoride (PMSF)
  • Protease and phosphatase inhibitors (Sigma‐Aldrich, cat. no. P8340 and P5726, respectively)
  • Bradford reagent (see recipe)
  • 6× Laemmli buffer (see recipe)
  • 10× SDS‐PAGE running buffer (see recipe)
  • Standard protein marker (e.g., Precision Plus Protein Dual Color Standard; BioRad, cat. no. 161‐0374)
  • Methanol
  • 10× transfer buffer (see recipe)
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A6003)
  • 10× Tris‐buffered saline/Tween 20 (TBST; see recipe)
  • Mouse monoclonal anti‐SHC/p66 – pSER36 (Calbiochem, cat. no. 566807)
  • Goat anti‐mouse IgG‐AP (Santa Cruz Biotechnology, cat. no. sc‐2008)
  • ECF substrate (Thermo Fisher Scientific, cat. no. RPN3685)
  • Mouse monoclonal anti‐SHC (BD Bioscience, cat. no. 610879)
  • Anti‐actin antibody, clone C4 (Millipore, cat. no. MAB1501)
  • 15‐ and 50‐ml disposable polystyrene tubes (e.g., BD Falcon)
  • 0.5‐ and 1.5 ml microcentrifuge tubes (e.g., VWR)
  • Accublock digital dry bath (LabNet international, Inc.)
  • Polyvinylidene difluoride (PVDF) membrane (Millipore, cat. no. IPVH00010) or nitrocellulose membrane (also see Gallagher et al., )
  • Mini Trans‐Blot Cell (BioRad, cat. no. 170‐3930)
  • Shaker plate
  • BioSpectrum multispectral imaging system (UVP)
  • Absorbance plate reader (e.g., Victor X3 Multilabel Reader, PerkinElmer)
  • Computer running Microsoft Excel, Quantity One 4.6.6 (BioRad), NIH ImageJ, and statistical software analysis
  • Additional reagents and equipment for protein assay ( appendix 3I; Krohn, ), SDS–polyacrylamide gel electrophoresis ( appendix 3F; Gallagher, ), immunoblotting (Gallagher et al., )

Alternate Protocol 1: Immunoblotting for Activity of p66Shc Pathway‐Associated Proteins

  Additional Materials (also see protocol 3)
  • Antibodies against p66Shc‐associated proteins of interest (Table 25.6.2)
Table 5.6.2   Additional Materials (also see protocol 3)Examples of Antibodies to Use in the Alternate Protocol

Protein/antibody against Vendor Catalog number
Bim Cell Signaling 2819
Forkhead box O3 (FoxO3a) Abcam ab4709
Forkhead box O3 (FoxO3a, phosphoSer253) Abcam ab47285
Pin1 Millipore 07‐091
Protein kinase B (PKB) or AKT (phosphoSer473) Cell Signaling 9271
Protein kinase B (PKB) or AKT Cell Signaling 9272
Protein kinase Cβ (PKCβ) Santa Cruz Biotechnology sc‐210
Protein phosphatase 2 (PP2A) Millipore 05‐421
Ras Cell Signaling 3965
Superoxide dismutase 1 (SOD 1) Santa Cruz Biotechnology sc‐11407
Superoxide dismutase 2 (SOD 2) Abcam ab13533

Support Protocol 1: Pharmacological Inhibition of p66Shc Pathway

  Materials
  • Cells of interest (e.g., H9c2 cardiomyoblasts, ATCC‐CRL‐1446)
  • High‐glucose DMEM (Sigma‐Aldrich, cat. no. D‐5648) containing 10% FBS (Invitrogen, cat. no. 16000‐044), 1× penicillin‐streptomycin (Invitrogen, cat. no. 15140‐122) and 1.5 g/liter sodium bicarbonate (Sigma‐Aldrich, cat. no. S5761)
  • Xenobiotic(s) of interest
  • 10‐cm2 cell culture dishes
  • 10 mM hispidin stock solution (see recipe)
  • Dimethyl sulfoxide (DMSO; Sigma‐Aldrich, cat. no. W387509)

Support Protocol 2: Knockdown of p66Shc in Cell Lines

  Materials
  • Cell line of interest
  • Appropriate medium for cell line of interest
  • p66Shc‐constructs (siRNA, shRNA)
  • Constructs with random sequence
  • Opti‐MEM medium (Invitrogen, cat. no. 11058‐021)
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668‐019)
  • 24‐well cell culture plates
  • 0.5 ml Eppendorf microcentrifuge test tubes (e.g., VWR)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Almeida, M., Han, L., Ambrogini, E., Bartell, S.M., and Manolagas, S.C. 2010. Oxidative stress stimulates apoptosis and activates NF‐kappaB in osteoblastic cells via a PKCbeta/p66shc signaling cascade: Counter regulation by estrogens or androgens. Mol. Endocrinol. 24:2030‐2037. doi: 10.1210/me.2010-0189.
  Arany, I., Clark, J., Reed, D.K., and Juncos, L.A. 2013a. Chronic nicotine exposure augments renal oxidative stress and injury through transcriptional activation of p66shc. Nephrol. Dial. Transplant. 28:1417‐1425. doi: 10.1093/ndt/gfs596.
  Arany, I., Clark, J.S., Reed, D.K., Juncos, L.A., and Dixit, M. 2013b. Role of p66shc in renal toxicity of oleic acid. Am. J. Nephrol. 38:226‐232. doi: 10.1159/000354357.
  Arany, I., Faisal, A., Clark, J.S., Vera, T., Baliga, R., and Nagamine, Y. 2010. p66SHC‐mediated mitochondrial dysfunction in renal proximal tubule cells during oxidative injury. Am. J. Physiol. Renal. Physiol. 298:F1214‐1221. doi: 10.1152/ajprenal.00639.2009.
  Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding. Anal. Biochem. 72:248‐254. doi: 10.1016/0003-2697(76)90527-3.
  Chen, Y.R., Han, J., Kori, R., Kong, A.N., and Tan, T.H. 2002. Phenylethyl isothiocyanate induces apoptotic signaling via suppressing phosphatase activity against c‐Jun N‐terminal kinase. J. Biol. Chem. 277:39334‐39342. doi: 10.1074/jbc.M202070200.
  Cigremis, Y., Turkoz, Y., Akgoz, M., and Sozmen, M. 2004. The effects of chronic exposure to ethanol and cigarette smoke on the level of reduced glutathione and malondialdehyde in rat kidney. Urol. Res. 32:213‐218. doi: 10.1007/s00240-004-0406-x.
  Clayton, D.A. and Shadel, G.S. 2014. Isolation of mitochondria from cells and tissues. Cold Spring Harb. Protoc. 2014:pdb top074542.
  Diogo, C.V., Suski, J.M., Lebiedzinska, M., Karkucinska‐Wieckowska, A., Wojtala, A., Pronicki, M., Duszynski, J., Pinton, P., Portincasa, P., Oliveira, P.J., and Wieckowski, M.R. 2013. Cardiac mitochondrial dysfunction during hyperglycemia–the role of oxidative stress and p66Shc signaling. Int. J. Biochem. Cell Biol. 45:114‐122. doi: 10.1016/j.biocel.2012.07.004.
  Gallagher, S.R. 2007. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Toxicol. 32:A.3F.1‐A.3F.38.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
  Gertz, M., Fischer, F., Wolters, D., and Steegborn, C. 2008. Activation of the lifespan regulator p66Shc through reversible disulfide bond formation. Proc. Natl. Acad. Sci. U.S.A. 105:5705‐5709. doi: 10.1073/pnas.0800691105.
  Hogeboom, G.H., Schneider, W.C., and Pallade, G.E. 1948. Cytochemical studies of mammalian tissues; isolation of intact mitochondria from rat liver: Some biochemical properties of mitochondria and submicroscopic particulate material. J. Biol. Chem. 172:619‐635.
  Ishola, D.A., Jr., Post, J.A., van Timmeren, M.M., Bakker, S.J., Goldschmeding, R., Koomans, H.A., Braam, B., and Joles, J.A. 2006. Albumin‐bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells. Kidney Int. 70:724‐731. doi: 10.1038/sj.ki.5001629.
  Koch, O.R., Fusco, S., Ranieri, S.C., Maulucci, G., Palozza, P., Larocca, L.M., Cravero, A.A., Farre, S.M., De Spirito, M., Galeotti, T., and Pani, G. 2008. Role of the life span determinant P66(shcA) in ethanol‐induced liver damage. Lab. Invest. 88:750‐760. doi: 10.1038/labinvest.2008.44.
  Kometiani, P., Liu, L., and Askari, A. 2005. Digitalis‐induced signaling by Na+/K+‐ATPase in human breast cancer cells. Mol. Pharmacol. 67:929‐936. doi: 10.1124/mol.104.007302.
  Krohn, R. I. 2005. The colorimetric detection and quantitation of total protein. Curr. Protoc. Toxicol. 23:A.3I.1‐A.3I.28.
  La Colla, A., Boland, R., and Vasconsuelo, A. 2015. 17beta‐estradiol abrogates apoptosis inhibiting PKCdelta, JNK and p66Shc activation in C2C12 Cells. J. Cell Biochem. 116(7):1454‐65.
  Le, S., Connors, T.J., and Maroney, A.C. 2001. c‐Jun N‐terminal kinase specifically phosphorylates p66ShcA at serine 36 in response to ultraviolet irradiation. J. Biol. Chem. 276:48332‐48336.
  Lebiedzinska, M., Duszynski, J., Rizzuto, R., Pinton, P., and Wieckowski, M.R. 2009. Age‐related changes in levels of p66Shc and serine 36‐phosphorylated p66Shc in organs and mouse tissues. Arch. Biochem. Biophys. 486:73‐80. doi: 10.1016/j.abb.2009.03.007.
  Lebiedzinska, M., Karkucinska‐Wieckowska, A., Giorgi, C., Karczmarewicz, E., Pronicka, E., Pinton, P., Duszynski, J., Pronicki, M., and Wieckowski, M.R. 2010. Oxidative stress‐dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders. Biochim. Biophys. Acta 1797:952‐960. doi: 10.1016/j.bbabio.2010.03.005.
  Lebiedzinska, M., Karkucinska‐Wieckowska, A., Wojtala, A., Suski, J.M., Szabadkai, G., Wilczynski, G., Wlodarczyk, J., Diogo, C.V., Oliveira, P.J., Tauber, J., Jezek, P., Pronicki, M., Duszynski, J., Pinton, P., and Wieckowski, M.R. 2013. Disrupted ATP synthase activity and mitochondrial hyperpolarisation‐dependent oxidative stress is associated with p66Shc phosphorylation in fibroblasts of NARP patients. Int. J. Biochem. Cell Biol. 45:141‐150. doi: 10.1016/j.biocel.2012.07.020.
  Lee, M.S., Igawa, T., Chen, S.J., Van Bemmel, D., Lin, J.S., Lin, F.F., Johansson, S.L., Christman, J.K., and Lin, M.F. 2004. p66Shc protein is upregulated by steroid hormones in hormone‐sensitive cancer cells and in primary prostate carcinomas. Int. J. Cancer 108:672‐678. doi: 10.1002/ijc.11621.
  Lee, S.K., Chung, J.I., Park, M.S., Joo, H.K., Lee, E.J., Cho, E.J., Park, J.B., Ryoo, S., Irani, K., and Jeon, B.H. 2011. Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C‐mediated p66shc phosphorylation and vasoconstriction. Cardiovasc. Res. 91:502‐509. doi: 10.1093/cvr/cvr095.
  Li, X., Xu, Z., Du, W., Zhang, Z., Wei, Y., Wang, H., Zhu, Z., Qin, L., Wang, L., Niu, Q., Zhao, X., Girard, L., Gong, Y., Ma, Z., Sun, B., Yao, Z., Minna, J.D., Terada, L.S., and Liu, Z. 2014. Aiolos promotes anchorage independence by silencing p66Shc transcription in cancer cells. Cancer Cell 25:575‐589. doi: 10.1016/j.ccr.2014.03.020.
  Lunghi, B., De Cunto, G., Cavarra, E., Fineschi, S., Bartalesi, B., Lungarella, G., and Lucattelli, M. 2015. Smoking p66Shc knocked out mice develop respiratory bronchiolitis with fibrosis but not emphysema. PLoS ONE 10:e0119797. doi: 10.1371/journal.pone.0119797.
  Luzi, L., Confalonieri, S., Di Fiore, P.P., and Pelicci, P.G. 2000. Evolution of Shc functions from nematode to human. Curr. Opin. Genet. Dev. 10:668‐674. doi: 10.1016/S0959-437X(00)00146-5.
  Magi, B. and Liberatori, S. 2005. Immunoblotting techniques. In Immunochemical Protocols, Vol. 295 (R. Burns, ed.) pp. 227‐253. Humana Press, Totowa, N.J.
  Marques‐Aleixo, I., Santos‐Alves, E., Mariani, D., Rizo‐Roca, D., Padrao, A.I., Rocha‐Rodrigues, S., Viscor, G., Torrella, J.R., Ferreira, R., Oliveira, P.J., Magalhaes, J., and Ascensao, A. 2015. Physical exercise prior and during treatment reduces sub‐chronic doxorubicin‐induced mitochondrial toxicity and oxidative stress. Mitochondrion 20:22‐33. doi: 10.1016/j.mito.2014.10.008.
  McConkey, D.J., Lin, Y., Nutt, L.K., Ozel, H.Z., and Newman, R.A. 2000. Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen‐independent, metastatic human prostate adenocarcinoma cells. Cancer Res. 60:3807‐3812.
  Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Reboldi, P., Pandolfi, P.P., Lanfrancone, L., and Pelicci, P.G. 1999. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402:309‐313. doi: 10.1038/46311.
  Migliaccio, E., Mele, S., Salcini, A.E., Pelicci, G., Lai, K.M., Superti‐Furga, G., Pawson, T., Di Fiore, P.P., Lanfrancone, L., and Pelicci, P.G. 1997. Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor‐MAP kinase‐fos signalling pathway. EMBO J. 16:706‐716. doi: 10.1093/emboj/16.4.706.
  Moore, C.B., Guthrie, E.H., Huang, M.T., and Taxman, D.J. 2010. Short hairpin RNA (shRNA): Design, delivery, and assessment of gene knockdown. Methods Mol. Biol. 629:141‐158.
  Muthukumaran, S., Sudheer, A.R., Menon, V.P., and Nalini, N. 2008. Protective effect of quercetin on nicotine‐induced prooxidant and antioxidant imbalance and DNA damage in Wistar rats. Toxicology 243:207‐215. doi: 10.1016/j.tox.2007.10.006.
  Nemoto, S. and Finkel, T. 2002. Redox regulation of forkhead proteins through a p66shc‐dependent signaling pathway. Science 295:2450‐2452. doi: 10.1126/science.1069004.
  Pani, G., Koch, O.R., and Galeotti, T. 2009. The p53‐p66shc‐Manganese Superoxide Dismutase (MnSOD) network: A mitochondrial intrigue to generate reactive oxygen species. Int. J. Biochem. Cell Biol. 41:1002‐1005. doi: 10.1016/j.biocel.2008.10.011.
  Pinton, P., Rimessi, A., Marchi, S., Orsini, F., Migliaccio, E., Giorgio, M., Contursi, C., Minucci, S., Mantovani, F., Wieckowski, M.R., Del Sal, G., Pelicci, P.G., and Rizzuto, R. 2007. Protein kinase C beta and prolyl isomerase 1 regulate mitochondrial effects of the life‐span determinant p66Shc. Science 315:659‐663. doi: 10.1126/science.1135380.
  Rao, D.D., Vorhies, J.S., Senzer, N., and Nemunaitis, J. 2009. siRNA vs. shRNA: Similarities and differences. Adv. Drug. Deliv. Rev.61:746‐759. doi: 10.1016/j.addr.2009.04.004.
  Ravichandran, K.S. 2001. Signaling via Shc family adapter proteins. Oncogene 20:6322‐6330. doi: 10.1038/sj.onc.1204776.
  Rosca, M.G., Vazquez, E.J., Chen, Q., Kerner, J., Kern, T.S., and Hoppel, C.L. 2012. Oxidation of fatty acids is the source of increased mitochondrial reactive oxygen species production in kidney cortical tubules in early diabetes. Diabetes 61:2074‐2083. doi: 10.2337/db11-1437.
  Savino, C., Pelicci, P., and Giorgio, M. 2013. The P66Shc/mitochondrial permeability transition pore pathway determines neurodegeneration. Oxid. Med. Cell. Longev. 2013:719407. doi: 10.1155/2013/719407.
  Schaffer, J.E. 2003. Lipotoxicity: When tissues overeat. Curr. Opin. Lipidol. 14:281‐287. doi: 10.1097/00041433-200306000-00008.
  Song, P., Yang, S., Xiao, L., Xu, X., Tang, C., Yang, Y., Ma, M., Zhu, J., Liu, F., and Sun, L. 2014. PKCdelta promotes high glucose induced renal tubular oxidative damage via regulating activation and translocation of p66Shc. Oxid. Med. Cell. Longev. 2014:746531.
  Sun, L., Xiao, L., Nie, J., Liu, F.Y., Ling, G.H., Zhu, X.J., Tang, W.B., Chen, W.C., Xia, Y.C., Zhan, M., Ma, M.M., Peng, Y.M., Liu, H., Liu, Y.H., and Kanwar, Y.S. 2010. p66Shc mediates high‐glucose and angiotensin II‐induced oxidative stress renal tubular injury via mitochondrial‐dependent apoptotic pathway. Am. J. Physiol. Renal Physiol. 299:F1014‐1025. doi: 10.1152/ajprenal.00414.2010.
  Suski, J.M., Karkucinska‐Wieckowska, A., Lebiedzinska, M., Giorgi, C., Szczepanowska, J., Szabadkai, G., Duszynski, J., Pronicki, M., Pinton, P., and Wieckowski, M.R. 2011. p66Shc aging protein in control of fibroblasts cell fate. Int. J. Mol. Sci. 12:5373‐5389. doi: 10.3390/ijms12085373.
  Tomita, K., Teratani, T., Suzuki, T., Oshikawa, T., Yokoyama, H., Shimamura, K., Nishiyama, K., Mataki, N., Irie, R., Minamino, T., Okada, Y., Kurihara, C., Ebinuma, H., Saito, H., Shimizu, I., Yoshida, Y., Hokari, R., Sugiyama, K., Hatsuse, K., Yamamoto, J., Kanai, T., Miura, S., and Hibi, T. 2012. p53/p66Shc‐mediated signaling contributes to the progression of non‐alcoholic steatohepatitis in humans and mice. J. Hepatol. 57:837‐843. doi: 10.1016/j.jhep.2012.05.013.
  Tuder, R.M., Kern, J.A., and Miller, Y.E. 2012. Senescence in chronic obstructive pulmonary disease. Proc. Am. Thorac. Soc. 9:62‐63. doi: 10.1513/pats.201201-012MS.
  Watabe, M., Masuda, Y., Nakajo, S., Yoshida, T., Kuroiwa, Y., and Nakaya, K. 1996. The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells. J. Biol. Chem. 271:14067‐14072. doi: 10.1074/jbc.271.41.25126.
  Wieckowski, M.R., Giorgi, C., Lebiedzinska, M., Duszynski, J., and Pinton, P. 2009. Isolation of mitochondria‐associated membranes and mitochondria from animal tissues and cells. Nat. Protoc. 4:1582‐1590. doi: 10.1038/nprot.2009.151.
  Wills, M.K. and Jones, N. 2012. Teaching an old dogma new tricks: Twenty years of Shc adaptor signalling. Biochem. J. 447:1‐16. doi: 10.1042/BJ20120769.
  Xiao, D. and Singh, S.V. 2002. Phenethyl isothiocyanate‐induced apoptosis in p53‐deficient PC‐3 human prostate cancer cell line is mediated by extracellular signal‐regulated kinases. Cancer Res. 62:3615‐3619.
  Xiao, D. and Singh, S.V. 2007. Phenethyl isothiocyanate inhibits angiogenesis in vitro and ex vivo. Cancer Res. 67:2239‐2246. doi: 10.1158/0008-5472.CAN-06-3645.
  Xiao, D. and Singh, S.V. 2010. p66Shc is indispensable for phenethyl isothiocyanate‐induced apoptosis in human prostate cancer cells. Cancer Res. 70:3150‐3158. doi: 10.1158/0008-5472.CAN-09-4451.
  Xiao, D., Johnson, C.S., Trump, D.L., and Singh, S.V. 2004. Proteasome‐mediated degradation of cell division cycle 25C and cyclin‐dependent kinase 1 in phenethyl isothiocyanate‐induced G2‐M‐phase cell cycle arrest in PC‐3 human prostate cancer cells. Mol. Cancer Ther. 3:567‐575.
  Xiao, D., Zeng, Y., Choi, S., Lew, K.L., Nelson, J.B., and Singh, S.V. 2005. Caspase‐dependent apoptosis induction by phenethyl isothiocyanate, a cruciferous vegetable‐derived cancer chemopreventive agent, is mediated by Bak and Bax. Clin. Cancer Res. 11:2670‐2679. doi: 10.1158/1078-0432.CCR-04-1545.
  Xie, Y. and Hung, M.C. 1996. p66Shc isoform down‐regulated and not required for HER‐2/neu signaling pathway in human breast cancer cell lines with HER‐2/neu overexpression. Biochem. Biophys. Res. Commun. 221:140‐145. doi: 10.1006/bbrc.1996.0559.
  Xu, C., Shen, G., Chen, C., Gelinas, C., and Kong, A.N. 2005. Suppression of NF‐kappaB and NF‐kappaB‐regulated gene expression by sulforaphane and PEITC through IkappaBalpha, IKK pathway in human prostate cancer PC‐3 cells. Oncogene 24:4486‐4495. doi: 10.1038/sj.onc.1208656.
  Yan, X., Liang, F., Li, D., and Zheng, J. 2015. Ouabain elicits human glioblastoma cells apoptosis by generating reactive oxygen species in ERK‐p66SHC‐dependent pathway. Mol. Cell. Biochem. 398:95‐104. doi: 10.1007/s11010-014-2208-y.
  Yang, C.P. and Horwitz, S.B. 2000. Taxol mediates serine phosphorylation of the 66‐kDa Shc isoform. Cancer Res. 60:5171‐5178.
  Yang, C.P. and Horwitz, S.B. 2002. Distinct mechanisms of taxol‐induced serine phosphorylation of the 66‐kDa Shc isoform in A549 and RAW 264.7 cells. Biochim. Biophys. Acta 1590:76‐83. doi: 10.1016/S0167-4889(02)00200-8.
  Zhou, S., Chen, H.Z., Wan, Y.Z., Zhang, Q.J., Wei, Y.S., Huang, S., Liu, J.J., Lu, Y.B., Zhang, Z.Q., Yang, R.F., Zhang, R., Cai, H., Liu, D.P., and Liang, C.C. 2011. Repression of P66Shc expression by SIRT1 contributes to the prevention of hyperglycemia‐induced endothelial dysfunction. Circ. Res. 109:639‐648. doi: 10.1161/CIRCRESAHA.111.243592.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library