Comment[ArrayExpressAccession]	E-GEOD-51266
MAGE-TAB Version	1.1						
Public Release Date	2013-10-01
Investigation Title	Gene expression changes induced by LKB1 expression in human lung adenocarcinoma cell lines						
Comment[Submitted Name]	Gene expression changes induced by LKB1 expression in human lung adenocarcinoma cell lines
Experiment Description	LKB1 is a tumor suppressor lost in approximately 30% of lung adenocarcinomas. It is a serine-threonine kinase involved in regulating metabolism, proliferation, and cell polarity. We have characterized its association with mRNA expression profiles in resected tumors and in cell lines, but little is known about the direct effects of LKB1 on the regulation of these genes. This study investigates the effects of LKB1 activity on mRNA expression in two LKB1-mutant lung adenocarcinoma cell lines, H2122 and A549. Wild-type LKB1 has been stably expressed in these cell lines using a pBABE retrovirus as well as an empty pBABE control and a kinase-dead mutant of LKB1 (K78I) control (Addgene). Samples submitted are two cell lines, three experimental conditions, and three replicates, for a total of 17 samples (one sample was excluded for poor RNA quality). Gene expression of these samples are analyzed to determine transcriptional regulatory effects of LKB1 expression. Results of this analysis are compared to our analysis of resected human tumors to determine gene patterns that are differentially expressed between LKB1-deficient and LKB1-wild-type tumors whose expression is also affected by restoration of LKB1 in vitro. RMA gene expression was taken from two cell lines stably expressing LKB1 or controls of K78I mutant LKB1 or empty pBABE vector. Log2 average expression differences are calculated and compared to results from analysis of gene expression associated with LKB1 loss in resected human tumors.						
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl					
Person Last Name	Carbone	David	Jacob				
Person First Name	David	Carbone	Kaufman				
Person Email	david.carbone@osumc.edu						
Person Affiliation	Ohio State University						
Person Address	Internal Medicine, Ohio State University, 460 West 12th Avenue; 488 BRT, Columbus, Ohio, USA						
Person Roles	submitter						
Protocol Name	P-GSE51266-1	P-GSE51266-5	P-GSE51266-6	P-GSE51266-2	P-GSE51266-3	P-GSE51266-4	P-GSE51266-7
Protocol Description	CEL files were analyzed on Affymetrix Expression Console v. 1.1 using a RMA normalization algorithm producing log base 2 results. ID_REF =  VALUE = Log2 RMA	Ambion WT Expression reactions for all samples were started using 130ng of total RNA. Resulting cRNA products from the overnight IVT reactions were cleaned with Ambion-WT bead cleanup kit. 10.5ug cRNA was used for cDNA synthesis. Resulting cDNA was cleaned with Ambion-WT bead cleanup kit. 5.5ug of cDNA was used for biotin labeling reactions.	5.5ug of fragmented, biotinylated cDNA were hybridized for 16 hours to a HT Human Gene 1.1 ST PM16 array plate utilizing the GeneTitan instrument.	Cell lines were stably transduced with pBABE retroviral constructs expressing wild-type LKB1 (LKB1-WT), or controls of empty pBABE (pBABE) or kinase dead mutant LKB1 (LKB1-K78I). Selection for cell lines with stable expression of these constructs was achieved by culturing cells in growth media containing 1.0 micrograms/milliliter puromycin for two weeks.	Cell lines were grown in RPMI1640 containing 5% fetal bovine serum, without antibiotics. Cells were harvested during logarithmic growth.	Trizol extraction of total RNA was performed according to the manufacturer's instructions. Isolated RNA was bioanalyzed using Agilent to determine 260/280 ratio, RNA concentration, and RIN.	HT Human Gene 1.1 ST chips were scanned using scanned on the Affymetrix Gene Titan AGCC v. 3.2.3
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	GENE TRANSDUCTION	CELL LINE					
Experimental Factor Type	gene transduction	cell line					
Comment[SecondaryAccession]	GSE51266						
Comment[GEOReleaseDate]	2013-10-01						
Comment[ArrayExpressSubmissionDate]	2013-09-30						
Comment[GEOLastUpdateDate]	2013-10-02						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-51266.sdrf.txt