Comment[ArrayExpressAccession] E-GEOD-34238 Public Release Date 2011-12-08 Investigation Title C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate Comment[Submitted Name] C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate Experiment Description In urodele amphibians, limb regeneration involves the dedifferentiation of muscle myotubes into single cells that may acquire pluripotent potential. We have employed small molecules (myoseverin and BIO) to attempt to reproduce this behavior in mammalian muscle culture. C2C12 myotubes derived from the C2C12 myoblast cell line were induced to undergo cellularization by myoseverin treatment, which destabilizes tubulin filaments. The GSK-3 inhibitor, BIO, was then used to induce dedifferentiation. Induce neuron formation; the cells were incubated with 250 nM reversine for 48 h, and neural induction media (DMEM/F12 supplemented with N2 (Invitrogen)) and 1.5 uM all-trans retinoic acid for 7 days. C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate (derived by 20 M myoseverin treatment for 48 h) C2C12 myoblasts were differentiated into myotubes with 2%horse serum in DMEM for 8 days (from 2-4 d, 10 uM AraC treatment was also used to kill any remaining myoblasts). Next, myotubes were cellularized by 20 uM myoseverin treatment for 48 h. 24 h after myoseverin treatment, myotubes were treated with 10 uM BIO for 2d. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Williams Williams Kim Person First Name Darren Darren Woong-hee Person Mid Initials Reece R Person Email darren@gist.ac.kr Person Affiliation Gwangju Institute of Science and Technology Person Phone 8210-8780-0915 Person Fax Person Address School of Life Sciences, Gwangju Institute of Science and Technology , 1 Oryong-dong, Gwangju , South Korea Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE34238-1 P-GSE34238-6 P-GSE34238-3 P-GSE34238-8 P-GSE34238-7 P-GSE34238-2 P-GSE34238-4 P-GSE34238-5 Protocol Description ID_REF =
VALUE = Quantile normalized signal intensity that passed QC After labeled aRNAs were placed on Agilent SurePrint G3 Human GE 8x60K array(Agilent technologies, CA) and covered by a Gasket 8-plex slide (Agilent technologies, CA). The slides were hybridized for 17hr at 65 ℃ oven. The hybridized slides were washed in 2 X SSC, 0.1 % SDS for 2 min, 1 X SSC for 3 min, and then 0.2 X SSC for 2 min at room temperature. The slides were centrifuged at 3000 rpm for 20 sec to dry. 1% antibiotics (penicillin and streptomycin) and 2% horse serum in DMEM is used for myotube formation and drug treatment. The data were processed based on quantile normalization method using the GeneSpring GX 11.5.1(Agilent technologies, CA). This normalization method aims to make the distribution of intensities for each array in a set of arrays the same. The normalized, and log transformed intensity values were then analyzed using GeneSpring GX 11.5.1 (Agilent technologies, CA). Fold change filters included the requirement that the genes be present in at least 200% of controls for up-regulated genes and lower than 50% of controls for down-regulated genes. The arrays were analyzed using an Agilent scanner with associated software. Gene expression levels were calculated with Feature Extraction v10.7.3.1 (Agilent technologies, CA) Relative signal intensities for each gene were generated using the Robust Multi-Array Average algorithm. In BIO treated cellulate; 30 uM myoseverine, 10 uM AraC and 10 uM BIO were treated in DMEM media containing 1% antibiotics (penicillin and streptomycin), 2% horse serum. In BIO+Neu treated cellulate; Cellulate was seeded in laminin-coated cultures dishes at a density of dishes at a density of 1.5x106 cells/9.6 cm². After small molecule treatment, the cells were incubated with 250 nM reversine for 48 h, and neural induction media (DMEM/F12 supplemented with N2 (Invitrogen)) and 1.5 uM all-trans retinoic acid for 7 days. RNA was prepared using the triazole solution. Extraction is followed isopropanol precipitation protocol: 1. wash with cold PBS. 2. Treat 1 mL trizaole/10 cm culture dish and stand 3min in room temperature. 3. Transfer reagent into a microtube. 4. Add chloroform (1/5 triazole solution), shake several times, and stand 15 min in room temperature. 5. centrifuge 12000 g for 10 min at 4℃ 6. Take transparent supernatent into a new microtube, add same volume of isopropanol and stand 5 min in room temperature. 7. Centrifuge 12000 g for 10 min at 4℃. 8. Remove supernatant and dry the RNA pellet. 8. Dissolve pellet with distilled water. Each total RNA sample (200ng) was labeled and amplified using Low Input Quick Amp labeling kit (Agilent technologies, CA). The Cy3-labeled aRNAs were resuspended in 50㎕ of hybridization solution (Agilent technologies, CA). Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization grow feature_extraction image_aquisition specified_biomaterial_action nucleic_acid_extraction labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name TREATMENT Experimental Factor Type treatment Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE34238 Comment[GEOLastUpdateDate] 2011-12-09 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2011-12-08 Comment[ArrayExpressSubmissionDate] 2011-12-07 SDRF File E-GEOD-34238.sdrf.txt