Bidirectional manipulation of SF-1 (NR5A1) in NCI-H295R cells

SF-1 (NR5A1) was overexpressed (Over) or knocked down with shRNA (shRNA) in NCI-H295R human adrenocortical tumor cells and differential global gene expression analysed 48 hours later using Affymetrix GeneChip Human Gene 1.0ST arrays. Over: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of a pIRES2-AcGPF1-Nuc construct co-expressing SF-1 cDNA and GFP. For experimental control, a mutagenized pIRES2 construct, bearing the G35E mutation in SF-1 that impairs its transactivation function in vivo and in vitro was used. shRNA: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of the SureSilencing shRNA Plasmid for Human NR5A1 with GFP marker kit (SABioscience). For experimental control, mismatch constructs provided in the kit were used. In both experiments (Over and shRNA), cells were harvested, prepared, and submitted to fluorescence-activated cell sorting (FACS) in a MoFlo XDP sorter 48 hours after transfection. Viable GFP-expressing cells were pooled and resuspended in TRIzol reagent for RNA extraction. Total RNA was extracted, and RNA quality control performed using a 2100 Bioanalyzer. RNA samples were processed using the Affymetrix GeneChip WT Sense Target Labeling kit, starting with 200 ng total RNA. Four independent Over experiments and five independent shRNA experiments were performed and samples of labeled fragmented cDNA were hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays. Based on quality control of array data (R/Bioconductor and Partek Genomics Suite), two Over arrays (one SF-1 wild type and one mutant control) and one shRNA array (mismatch control) were excluded and are not deposited here. Differential gene expression analysis was performed using the limma package in R/Bioconductor. A Benjamini-Hochberg-corrected P value cut-off of 0.05 was used to select significant differentially expressed genes in each setting (Over and shRNA). Finally, results from both analyses were combined to identify a subset of positively-regulated SF-1 target genes in these cells, ie transcripts up-regulated by SF-1 overexpression (Over) and down-regulated by SF-1 knockdown (shRNA). Four SF-1 overexpression experiments (Over; intervention = overexpression of wild type SF-1; control = overexpression of functionally-impaired mutant G35E SF-1) and five SF-1 knockdown experiments (shRNA; intervention = SF-1-specific small hairpin RNA; control = shRNA mismatch control) were performed in NCI-H295R cells. Differential gene expression in each setting was analysed using GeneChip Human Gene 1.0ST arrays. Data for arrays that met quality control are presented here: Over, 3 intervention and 3 control arrays; shRNA, 5 intervention and 4 control arrays. Total of 15 arrays.