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• W2169179975 abstract "Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122–6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers. Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122–6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers. MLN4924 is an investigational small molecule inhibitor of the NEDD8-activating enzyme (NAE) 1The abbreviations used are: NAENedd8-activating enzymeCRLcullin-RING ubiquitin ligaseRNAiRNA interferenceBIBliss independenceSILACStable isotope labeling by amino acids in cell cultureDMEMDulbecco's modified Eagle's mediumDMSOdimethylsulfoxideRIPAradioimmunoprecipitation assay. 1The abbreviations used are: NAENedd8-activating enzymeCRLcullin-RING ubiquitin ligaseRNAiRNA interferenceBIBliss independenceSILACStable isotope labeling by amino acids in cell cultureDMEMDulbecco's modified Eagle's mediumDMSOdimethylsulfoxideRIPAradioimmunoprecipitation assay. (1Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1351) Google Scholar) that is currently being explored in Phase I clinical trials. MLN4924 has been shown to be a selective inhibitor of NAE, inhibiting ∼9% of bulk protein turnover in cells without impacting protein synthesis (1Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1351) Google Scholar). Inhibition of NAE leads to the stabilization of a minor subset of proteasome-degraded proteins, namely those ubiquitinated in a cullin-RING ligase (CRL) dependent fashion (1Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1351) Google Scholar). Many of the proteins targeted by cullins are known to have cancer relevance (2Guardavaccaro D. Pagano M. Oncogenic aberrations of cullin-dependent ubiquitin ligases.Oncogene. 2004; 23: 2037-2049Crossref PubMed Scopus (72) Google Scholar, 3Soucy T.A. Dick L.R. Smith P.G. Milhollen M.A. Brownell J.E. The NEDD8 conjugation pathway and its relevance in cancer biology and therapy.Genes Cancer. 2010; 1: 708-716Crossref PubMed Scopus (166) Google Scholar, 4Lee J. Zhou P. Cullins and cancer.Genes Cancer. 2010; 1: 690-699Crossref PubMed Scopus (67) Google Scholar). In particular, the stabilization of Cdt1 leads to DNA rereplication and accumulation of cells in S-phase and this effect has been shown to be especially important for cell death by MLN4924 in most cancer cell lines studied (1Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1351) Google Scholar, 5Lin J.J. Milhollen M.A. Smith P.G. Narayanan U. Dutta A. NEDD8-targeting drug MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells.Cancer Res. 2010; 70: 10310-10320Crossref PubMed Scopus (219) Google Scholar, 6Milhollen M.A. Narayanan U. Soucy T.A. Veiby P.O. Smith P.G. Amidon B. Inhibition of NEDD8-activating enzyme induces re-replication and apoptosis in human tumor cells by deregulating CDT1 turnover.Cancer Res. 2011; 71: 3042-3051Crossref PubMed Scopus (122) Google Scholar), although stabilization of IκB plays a role in some settings (7Milhollen M.A. Traore T. Adams-Duffy J. Thomas M.P. Berger A.J. Dang L. Dick L.R. Garnsey J.J. Koenig E. Langston S.P. Manfredi M. Narayanan U. Rolfe M. Staudt L.M. Soucy T.A. Yu J. Zhang J. Bolen J.B. Smith P.G. MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-{kappa}B-dependent lymphoma.Blood. 2010; 116: 1515-1523Crossref PubMed Scopus (261) Google Scholar). Rereplication leads to the activation of DNA damage repair processes, including ATR and ATM. However, it is likely that additional proteins affecting the sensitivity of cancer cells are stabilized by MLN4924. Such proteins may include NFE2L2 (Nrf2), p21, p27, cyclin E1, cyclin D1, Emi1, and Orc1, all of which are previously characterized CRL substrates (6Milhollen M.A. Narayanan U. Soucy T.A. Veiby P.O. Smith P.G. Amidon B. Inhibition of NEDD8-activating enzyme induces re-replication and apoptosis in human tumor cells by deregulating CDT1 turnover.Cancer Res. 2011; 71: 3042-3051Crossref PubMed Scopus (122) Google Scholar). The identification of proteins that are stabilized by MLN4924 and the impact they have on cell death could provide important insights into the mechanism of cell death, inform the clinical utility of MLN4924, and identify possible pharmacodynamic and predictive biomarkers. It would also expand our understanding of the biological roles of the cullins. Nedd8-activating enzyme cullin-RING ubiquitin ligase RNA interference Bliss independence Stable isotope labeling by amino acids in cell culture Dulbecco's modified Eagle's medium dimethylsulfoxide radioimmunoprecipitation assay. Nedd8-activating enzyme cullin-RING ubiquitin ligase RNA interference Bliss independence Stable isotope labeling by amino acids in cell culture Dulbecco's modified Eagle's medium dimethylsulfoxide radioimmunoprecipitation assay. The NEDD8-activating enzyme transfers the small ubiquitin-like protein NEDD8 onto Ubc12 in an ATP-dependent fashion, which then transfers NEDD8 onto one of seven cullins (8Merlet J. Burger J. Gomes J.E. Pintard L. Regulation of cullin-RING E3 ubiquitin-ligases by neddylation and dimerization.Cell. Mol. Life Sci. 2009; 66: 1924-1938Crossref PubMed Scopus (139) Google Scholar). Cullins are subunits within the CRL family of ubiquitin E3 ligases. Neddylation of the cullin allows the associated ubiquitin E2 enzyme to polyubiquitinate its substrate, thereby targeting it to the proteasome for degradation (9Duda D.M. Borg L.A. Scott D.C. Hunt H.W. Hammel M. Schulman B.A. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation.Cell. 2008; 134: 995-1006Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). Additional proteins modified by NEDD8 have been proposed (10Xirodimas D.P. Novel substrates and functions for the ubiquitin-like molecule NEDD8.Biochem. Soc. Trans. 2008; 36: 802-806Crossref PubMed Scopus (148) Google Scholar, 11Xirodimas D.P. Sundqvist A. Nakamura A. Shen L. Botting C. Hay R.T. Ribosomal proteins are targets for the NEDD8 pathway.EMBO Rep. 2008; 9: 280-286Crossref PubMed Scopus (137) Google Scholar), as have proteins that associate with NEDD8 (12Jones J. Wu K. Yang Y. Guerrero C. Nillegoda N. Pan Z.Q. Huang L. A targeted proteomic analysis of the ubiquitin-like modifier nedd8 and associated proteins.J. Proteome Res. 2008; 7: 1274-1287Crossref PubMed Scopus (128) Google Scholar, 13Norman J.A. Shiekhattar R. Analysis of Nedd8-associated polypeptides: a model for deciphering the pathway for ubiquitin-like modifications.Biochemistry. 2006; 45: 3014-3019Crossref PubMed Scopus (16) Google Scholar). The dynamics of the cullin interactome following inhibition of NAE by MLN4924 has recently been extensively studied (14Bennett E.J. Rush J. Gygi S.P. Harper J.W. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.Cell. 2010; 143: 951-965Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Proteomic experiments designed to identify ubiquitinated proteins have primarily used epitope-tagged ubiquitin (15Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1307) Google Scholar, 16Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.Mol. Cell. Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 17Gururaja T. Li W. Noble W.S. Payan D.G. Anderson D.C. Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing.J. Proteome Res. 2003; 2: 394-404Crossref PubMed Scopus (35) Google Scholar, 18Hitchcock A.L. Auld K. Gygi S.P. Silver P.A. A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12735-12740Crossref PubMed Scopus (134) Google Scholar, 19Bazile F. Gagné J.P. Mercier G. Lo K.S. Pascal A. Vasilescu J. Figeys D. Poirier G.G. Kubiak J.Z. Chesnel F. Differential proteomic screen to evidence proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryos.J. Proteome Res. 2008; 7: 4701-4714Crossref PubMed Scopus (3) Google Scholar, 20Mayor T. Lipford J.R. Graumann J. Smith G.T. Deshaies R.J. Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.Mol. Cell. Proteomics. 2005; 4: 741-751Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 21Franco M. 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However, because NAE inhibition blocks the ubiquitination of a minor subset of proteasome substrates, approaches relying on changes in global ubiquitination are unlikely to sufficiently enrich NAE-dependent changes. Recently, major strides in the identification and quantification of proteins by mass spectrometry have been achieved by improvements in methodology and instrumentation. Stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a particularly promising approach to quantitate protein abundance. A number of recent studies that provide a global quantitation of proteins from cell extracts have identified between 3880 and 5619 proteins (28Walther D.M. Mann M. Accurate quantification of more than 4,000 mouse tissue proteins reveals minimal proteome changes during aging.Mol. Cell. Proteomics. 2011; 10 (M110.004523)Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 29Graumann J. Hubner N.C. Kim J.B. Ko K. Moser M. Kumar C. Cox J. Schöler H. Mann M. Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins.Mol. Cell. Proteomics. 2008; 7: 672-683Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 30de Godoy L.M. Olsen J.V. Cox J. Nielsen M.L. Hubner N.C. Fröhlich F. Walther T.C. Mann M. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.Nature. 2008; 455: 1251-1254Crossref PubMed Scopus (737) Google Scholar, 31Bonaldi T. Straub T. Cox J. Kumar C. Becker P.B. Mann M. Combined use of RNAi and quantitative proteomics to study gene function in Drosophila.Mol. Cell. 2008; 31: 762-772Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 32Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. 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Herein, we detail our global quantitation by SILAC of proteins within A375 melanoma cells treated with MLN4924 or aphidicolin, an inhibitor of S-phase. We identified 7689 proteins with two or more unique peptides in at least one sample. One hundred and thirty proteins were confidently up-regulated by MLN4924 by 1.8-fold or greater; 29 of 30 proteins evaluated by Western blotting were confirmed. Many of the proteins identified as being up-regulated by MLN4924 were near detection limits of the mass spectrometer. Furthermore, a larger set of 606 up-regulated proteins were identified by relaxing the selection criteria. Their impact on MLN4924-sensitive biology was then assessed by evaluating their genetic interaction with MLN4924 on cell viability by RNA interference. Thirty-eight of these stabilized proteins gave significant genetic interactions with MLN4924, half of which were confidently up-regulated. Four of 16 proteins identified with less confident mass spectrometric data but with demonstrated genetic interactions were confirmed to be up-regulated at least 1.8-fold by Western blot analysis. Strikingly, MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 were stabilized within 4 h, suggesting that these may be novel CRL substrates. These results suggest that the sensitivity of SILAC experiments using modern analytical techniques has now made near proteome-wide analysis of ubiquitin-proteasome system achievable. A375 cells were grown for 11 days with three passages in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin and containing either 0.5 mm each of l-Lysine-2HCl and l-Arginine-HCl or 13C615N2 l-Lysine-2HCl and 13C615N4 l-Arginine-HCl. All reagents for isotope metabolic protein labeling of the cells were from Pierce (Rockford, IL) except that l-glutamine and penicillin/streptomycin were from Invitrogen (Carlsbad, CA). A sample of 1.5 × 106 cells from the “light” and “heavy” isotope protein-labeled populations were plated separately in 10-cm dishes and treated with 650 nm MLN4924 or dimethyl sulfoxide (DMSO) (0.05% v/v final concentration) for 1, 4, or 24 h. Cells were scraped into ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 50 mm sodium fluoride, 25 mm β-glycerophosphate, 50 mm sodium orthovanadate, 10 mm iodoacetamide, 1,10-phenanthroline monohydrate and 500 U/ml benzonase nuclease HC. Lysates were combined such that for each time point, drug-treated heavy metabolically labeled cells were mixed 1:1 by cell count with vehicle-treated light metabolically labeled cells, and vice versa. Lysates were stored frozen overnight at −80 °C. Following thawing of the sample, the soluble fraction was prepared by centrifugation of the extracts at 14,000 rpm for 10 min at 4 °C. Samples were mixed with 2 × Laemmli SDS sample buffer containing alkylation reagent iodoacetamide (final concentration 50 mm). After 30 min of incubation at room temperature in the dark, the lysates were fractionated on a large format Protean II xi (BioRad, Hercules, CA) tris-glycine SDS-PAGE gel (8–16%). The gel was sliced into 70–80 sections and tryptic in-gel digestion of the gel slices was performed (36Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3531) Google Scholar). Dried protein digests were reconstituted in a solution containing 1% formic acid and 2% acetonitrile (v/v) for liquid chromatography/tandem MS (LC/MS/MS) analysis. Reconstituted protein digests were analyzed on a LC/MS/MS system comprised of an Eksigent NanoLC Ultra 2D-Plus liquid chromatography system, a NanoLC AS-2 autosampler (Eksigent, Dublin, CA), and a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A 15 cm PicoFrit column (75 μm inner diameter) packed with ProteoPep II C18 packing material (New Objective, Woburn, MA) was used for online peptide separation. A linear gradient of 2–40% B (89.9% acetonitrile and 0.1% formic acid with A as 0.1% formic acid) was used to elute peptides over 85 min. Eluted peptides were sprayed into the mass spectrometer through a Digital PicoView nanospray ion source (New Objective, Woburn, MA). MS1 scans were acquired using an AGC target of 2 × 106 for the Orbitrap, and a resolution of 100,000 at 400 amu. Each MS1 scans was followed by 10 data-dependent collision-induced dissociation MS/MS scans in the iontrap. Raw MS data were processed using a combination of MaxQuant (version 1.0.13.13) (32Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar) and Mascot (version 2.2.03, Matrix Science, London, UK). MaxQuant was used for postacquisition precursor m/z calibration, MS/MS spectrum peak picking, SILAC peptide ratio calculation, as well as protein grouping and quantitation. Database searching was done by using Mascot. For peptide/protein identification, the following modifications were included: carbamidomethylation (Cys, fixed), oxidization (Met, variable), N-acetylation (protein, variable), and pyro (Gln, variable). Trypsin specificity was set to exclude cleavages between Lys-Pro and Arg-Pro. Up to two missed cleavages and three labeled SILAC residues (Lys and Arg) per peptide were allowed. The MS/MS data were searched against a catenated database combining the International Protein Index (IPI) human database version 3.63 (84,118 sequences), the reversed sequences of all sequences in the IPI human database, and 262 commonly observed contaminates. The global false discovery rate for both peptides and proteins were set to 0.01. The posterior error probability threshold for peptide identification was set to 1 (no filtering). Minimum peptide length was six residues. Mass tolerances for precursor ions and product ions were 7 ppm (after calibration by MaxQuant) and 0.5 Da, respectively. For protein quantitation, three modifications were included, carbamidomethylation (Cys), oxidization (Met), and pyro (Gln), along with all unmodified peptides. Requantify function was enabled to capture SILAC pairs that were missed in the initial SILAC pair identification. A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Three hundred cells/well were reverse transfected with 15 nm siRNA (siGENOME SMARTpool, Dharmacon) in Optimem (Invitrogen) using 20 nL/well DharmaFECT 4 transfection reagent (Dharmacon, Lafayette, CO). Transfection occurred in 384-well CELLCOAT poly-d-lysine coated, black, clear-bottom plates (Greiner, Longwood, FL). Following 48 h incubation, 250 nm MLN4924, 650 nm MLN4924, or 0.27% DMSO was added. After an additional 48 h incubation, viability was assayed using ATPlite (Perkin Elmer) and luminescence was measured using a LeadSeeker plate reader (GE Healthcare). SMARTpool deconvolution was performed identically, except that individual duplexes were transfected at 8 nm. A375 cells were seeded on 6-well plates (Falcon #353046) with 5 × 105 cells/well (2 ml media) at 37 °C overnight. Following treatment with 650 nm MLN4924 or vehicle (0.065% DMSO) for the times indicated, A375 cells were harvested by trypsinization and centrifugation and stored in 350 μl RLT (Qiagen #79216) and 2-mercaptoethanol at −80 °C. Total RNA was extracted using MagMAX kits (Ambion, Austin, TX; #AM1839) on KingFisher Magnetic Particle Processors (Thermo Scientific). Labeled antisense RNA (cRNA) was synthesized by using the MessageAmp™ Premier RNA Amplification Kit (Ambion #AM1792). The resulting cRNA was then evaluated on Affymetrix Human Genome U133 Plus 2.0 arrays. All procedures were done according to manufacturers' protocols and specifications. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE30531 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE30531). Triplicate results were normalized by RMA and scored for significance by 2-way ANOVA for drug-time interaction with Benjamini-Hochberg multiple test correction (FDR) p < 0.05 being judged as significant. Sum of differences between MLN4924 and vehicle treatment were used to rank effect sizes. A375 cells were maintained at 37 °C in a humidified atmosphere of 6% CO2:94% air using DMEM medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and 100 U/ml penicillin, 100 g/ml streptomycin (Invitrogen). Following treatment with 650 nm MLN4924 or vehicle (0.065% DMSO) for the times indicated, cells were rinsed once with phosphate-buffered saline (PBS) and then harvested by scraping and centrifugation. Cell pellets were lysed in RIPA buffer containing 1 × protease inhibitor mixture (Calbiochem, #539131), 50 mm sodium fluoride, 50 mm sodium orthovanadate, 25 mm β-glycerophosphate, and benzonase, followed by freezing at −80 °C overnight and thawing at 4 °C with periodic vortexing. Insoluble material was removed by centrifugation (Eppendorf refrigerated microfuge, 14,000 rpm for 20 min). Protein concentration was determined by Bradford Assay (Pierce) and samples were adjusted with RIPA buffer and normalized with tubulin on quantitative immunoblots (Odyssey Infrared Imager, LI-COR Biosciences, Lincoln, NE) so that the coefficient of variance across samples was <16%. Samples were loaded on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and electrophoresed in MES/SDS buffer (Invitrogen), or NuPAGE 3–8% Tris-Acetate gels and electrophoresed in Tris-Acetate SDS running buffer (Invitrogen) for large molecular weight proteins. Proteins were transferred to Immobilin FL PVDF membrane (Millipore) by the semi-dry transfer method. Membranes were blocked for 1 h in Odyssey Blocking buffer (LI-COR Biosciences) and then were incubated with primary antibodies (supplemental Table S7) in Odyssey Blocking buffer/Tris-buffered saline, 0.1% Tween 20 (TBST) overnight at 0–4 °C. After washing three times in TBST, membranes were incubated with the appropriate Alexa Fluor 680 secondary antibody in Odyssey Blocking buffer/TBST at indicated dilutions for 1 h at room temperature. Following five washes in TBST and one wash in TBS, membranes were dried for 1 h at room temperature and proteins were detected and quantitated by LI-COR/Odyssey infrared image system (LI-COR Biosciences). Relative induction (Irel) of proteins was calculated as follows: lrel=(ld,t-lv,2)/(max(lM,t)-lv,2)Eq. 1 Where Id,t is the LI-COR intensity with treatment = d at time = t; Iv,2 is LI-COR intensity with vehicle treatment at time = 2 h; and IM,t is LI-COR intensity with MLN4924 treatment at time = t. For supplemental Fig. S6, Iv,t was used instead of IM,t. As an inhibitor of the NAE, the primary effect of MLN4924 is the stabilization of proteins rapidly turned over by the CRLs. In order to obtain a more complete understanding of the effects of this inhibition, A375 melanoma cells were prepared for SILAC analysis" @default.
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• W2169179975 title "Quantitative Proteomic Analysis of Cellular Protein Modulation upon Inhibition of the NEDD8-Activating Enzyme by MLN4924" @default.
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