Mors: RCC22mut and RCC243mut have gain of chromosome 7, RCC243mut also has loss of chromosome 4, and RCC364mut has 2 deletions, one in chromosome 8 and one in chromosome 9. In comparison, 786? and A-498 had considerably more CNAs than primary ccRCCs. We performed RNAseq on 6 VHLmut/VHLwt cell culture pairs grown in 10 FBS at passage 2 to 5, as well as RNA extracted from the matched primary tumorLobo et al. BMC Cancer (2016) 16:Page 8 ofFig. 4 Copy number profiles of VHLmut and VHLwt cultures and their matched tissues. Three VHLmut cultures that had reached passage 20 were profiled using Illumina Human Omni2.5? SNP arrays. DNA samples obtained from the matching primary tumor and adjacent normal kidney tissue were profiled in parallel, as well as two commercially available cell lines, 786? and A-498. For RCC22 and RCC243, the matched VHLwt line was also profiled; RCC364 does not have a matched VHLwt line. Amplifications are shown in blue and deletions in red. VHLwt cell lines do not display any gross genomic alterations, whereas VHLmut cell lines contain alterations matching the tumors from which they were derived. 786? and A-498 (bottom), have considerably larger numbers of alterations compared to primary tumors and RCC VHLmut culturestissues. RNAseq data has been deposited in NCBI’s Gene Expression Omnibus [21] and are accessible through GEO Series accession number GSE74958. Upon unsupervised clustering of gene expression profiles the tumor tissues clustered discretely from the cultured cells, and the VHLmut and VHLwt cultures formed discrete subclusters, indicating distinct expression profiles within each sample type (Additional file 10: Figure S5). 593 differentially Ixazomib citrateMedChemExpress MLN9708 expressed protein-coding genes (cut-off of 2-fold and adjusted p-value 0.01) were identified between VHLmut and VHLwt cell cultures (Fig. 5a). Genes differentially expressed in VHLmut cells included hypoxia response genes and genes related to metabolism, as expected (Additional file 5: Table S5, Additional file 10: Figure S6). Gene set enrichment analysis (GSEA) of the VHLmut and VHLwt gene signatures against RNAseq data from patient-matched primary tumor and adjacent normal tissues generated by The Cancer Genome Atlas (TCGA) [7] showed significant enrichment of the VHLmut signature in ccRCC tumor tissues, and of the VHLwt signature in normal adjacent kidney tissues, respectively (Fig. 5b). To gain a better understanding of differentially expressed networks in VHLmut vs. VHLwt cells, we PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 also performed GSEA of a compiled database of gene sets relating to biological processes, molecular functions and pathways [22] against a ranked gene list of our entire expression dataset [23]. Results were visualized using Cytoscape with the Enrichment Map plugin [22]. Only a fewnetworks were up-regulated in VHLmut cells, including glycolysis and electron transport, hypoxia/oxygen response, epigenetics/histone modification and bile acid and bile salt transport (Fig. 6, Additional file 6: Table S6). The top differentially expressed genes associated with each of these networks are listed in Additional file 7: Table S7. A larger number of pathways and networks were enriched in VHLwt cells, including cell-cell and cellmatrix interactions, focal adhesion and cytoskeleton organization, epithelial and endothelial differentiation, growth factor pathways such as TGF and TNF, glycosylation, and RNA processing and transport (Additional file 6: Table S6). An analysis of differentially expressed gen.