Comment[ArrayExpressAccession]	E-GEOD-29909
Public Release Date	2011-08-31										
Investigation Title	Modeling OPMD in myotube cultures reveals reduced accumulation of soluble mutant PABPN1 protein										
Comment[Submitted Name]	Modeling OPMD in myotube cultures reveals reduced accumulation of soluble mutant PABPN1 protein
Experiment Description	Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in Poly(A)-binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. The transgene expression is induced upon myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to the in vivo aggregates. Quantitative analysis of PABPN1 protein in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. In a comparative study we found that aggregation of expPABPN1 is more affected by inhibition of proteasome activity, as compared with the WT PABPN1 aggregation. Consistent with this, in myotubes cultures expressing expPABPN1 deregulation of the proteasome was identified as the most significantly deregulated pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and protein turnover. This study indicates, for the first time, that in myotubes the ratio of soluble to insoluble expPABPN1 is significantly lower compared to that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD. Clones on IM2 mouse myotubes that stably express Ala10-PABPN1-FLAG (WTA, WTD) or Ala17-PABPN1-FLAG (D7E). The transgene expression level in D7E and WTA are similar. WTA and WTD reflects differences in expression levels. RNA was extracted from myotubes of WTA, WTD and D7E in triplicates. cDNA synthesis and lebeling was preformed with the Illumina cDNA labeling kit.										
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Term Source File	http://www.ebi.ac.uk/efo/efo.owl										
Person Last Name	Anvar	Raz	Routledge	Venema	Buijze	van der Wal	Yahya Anvar	Straasheijm	Klooster	Antoniou	van der Maarel
Person First Name	Seyed Yahya	Vered	Samantha	Andrea	Hellen	Erik	Seyed	Kirsten	Rinse	Michael	Silvere
Person Mid Initials								R			M
Person Email	s.y.anvar@lumc.nl										
Person Affiliation	Leiden University Medical Center										
Person Phone	0031715269489										
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Person Address	Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, Leiden, Netherlands										
Person Roles	submitter										
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Protocol Name	P-GSE29909-1	P-GSE29909-6	P-GSE29909-3	P-GSE29909-8	P-GSE29909-7	P-GSE29909-2	P-GSE29909-4	P-GSE29909-5			
Protocol Description	ID_REF = <br>VALUE = quantile-normalized signal	Total RNA was extracted from myotubes using RNA Bee (Amsbio) according to the manufacturer's instructions. RNA integration number (RIN) was determined with RNA 6000 Nano (Agilent Technologies). RNA with RIN >7 were used for subsequent steps. RNA labeling was performed with the Illumina® TotalPrep RNA Amplification kit (Ambion) according to the manufacturer's protocol, and subsequently was hybridized to Illumina Mouse v2 Bead arrays.	Myoblasts were seeds to confluence and incubated at fusion conditions for 4 days, followed by RNA extraction	Microarray measurements were normalized using the quantile method. The quality of the data was assessed by principal component analysis.	Standard Illumina protocol	No treatments	RNA Bee (Amsbio) according to the manufacturer's instructions.	Illumina® TotalPrep™ RNA Amplification Kit  AMIL1791			
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Protocol Type	bioassay_data_transformation	hybridization	grow	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic_acid_extraction	labeling			
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Experimental Factor Name	GENOTYPE/VARIATION										
Experimental Factor Type	genotype/variation										
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Comment[SecondaryAccession]	GSE29909										
Comment[GEOLastUpdateDate]	2011-08-31
Comment[AEExperimentType]	transcription profiling by array										
Comment[GEOReleaseDate]	2011-08-30
Comment[ArrayExpressSubmissionDate]	2011-06-12
SDRF File	E-GEOD-29909.sdrf.txt