in addition, when looking at differentially expressed genes unique to certain comparisons, the largest group of genes were found common to all pairwise comparisons between hek293 and each of the progeny cell lines ( green bar in fig. 3b ), again emphasizing a relatively high degree of common transcriptomic changes amongst progeny cell lines differentiated from the parental hek293. as the progeny cell lines had an enrichment of differentially expressed genes associated with cellular component organization compared to hek293, we sought to evaluate to what cellular compartments the 329 genes common to all pairwise comparisons between hek293 and progeny cell lines localize. in line with the overall differential expression evaluation ( fig. 3a ), which emphasized changes in for instance cell adhesion and extracellular matrix organization, there was a significant ( padj < 0. 05 ) enrichment of genes relating to the integral compartment of plasma membrane ( go: 0005887 ) amongst the common differentially expressed ( de ) genes unique to the comparisons between hek293 and all progeny cell lines ( fig. 3c ). moreover, gene set analysis of these 329 genes showed, although non - significant in this limited set of genes, alterations in processes related to cell surface, cell adhesion and epithelial to mesenchymal transition ( supplementary fig. s6 ). the growth morphology of bioproduction cell lines is of great importance for culture maintenance and efficiency of industrial bioprocessing. in order to look into gene expression variations correlating with adherent and suspension hek 293 cell lines, differential expression analysis between adherent and suspension hek293 progeny cell lines was performed. as results from the overall comparison of transcriptomic profiles of the hek293 cell lines showed that the parental hek293 cell line is highly differentiated from all of the progeny cell lines and moreover, that the freestyle 293 - f cell line is very similar to the 293 - f cell line, hek293 and freestyle 293 - f were excluded from this analysis, so as not to skew the data. enrichment analysis of the differentially expressed genes between adherent ( 293t and 293e ) and suspension ( 293 - h and 293 - f ) progeny cell lines showed significant expression differences of similar gene sets as in the comparison between progeny cell lines and the parental hek293 ( figs. 3a, 4a, supplementary table s6 ). for instance, the suspension progeny cell lines had a significant up - regulation of gene sets involved in cellular compartment organization such as cell morphogenesis, cell junction -, cell membrane - and cytoskeleton organization. interestingly, there is no significant change in the expression of the extracellular matrix organization gene set between suspension and adherent hek293 progeny cell lines. perhaps as expected, there was significant differential expression observed for the cell adhesion, cell differentiation, cell morphogenesis and cell motility gene sets. all of the above - mentioned gene sets, including cell adhesion, were up - regulated in suspension hek293 cells as compared to adherent. when looking at the most significantly differentially expressed genes ( adjusted p - value < 0. 01 ) amongst the cell adhesion gene set, many genes of the cadherin superfamily of cell adhesion molecules were found up - regulated in suspension cell lines compared to adherent hek293 progeny cells ( supplementary table s7 ). figure 4gene set analysis identified biological process and metabolic changes between suspension and adherent hek293 progeny cell lineages. ( heatmap of gene set analysis using go biological processes and piano consensus scores showed a different expression pattern between suspension cells ( 293 - h and 293 - f ) and adherent cells ( 293e and 293t ). low consensus scores, represented by a dark blue color, indicate more significant differences. ( metabolic genes set analysis for comparing metabolic differences between suspension and adherent hek293 progeny cell lines. the size of each node corresponds to the number of genes in each of these pathways, thickness of connections between nodes corresponds to the number shared genes between pathways and the colors of the nodes shows the p - value for the given metabolic process. gene set analysis identified biological process and metabolic changes between suspension and adherent hek293 progeny cell lineages. ( heatmap of gene set analysis using go biological processes and piano consensus scores showed a different expression pattern between suspension cells ( 293 - h and 293 - f ) and adherent cells ( 293e and 293t ). low consensus scores, represented by a dark blue color, indicate more significant differences. ( metabolic genes set analysis for comparing metabolic differences between suspension and adherent hek293 progeny cell lines. the size of each node corresponds to the number of genes in each of these pathways, thickness of connections between nodes corresponds to the number shared genes between pathways and the colors of the nodes shows the p - value for the given metabolic process. in order to evaluate what metabolic impact the differentially expressed genes may have on cells in suspension compared to the adherent state, a generic human metabolic model, hmr2, was used to generate a set of metabolic genes and their assigned pathways to find metabolic pathways with altered expression between adherent and suspension hek293 progeny cell lines. as shown in fig. 4b, pathways related to aromatic amino acids and oxidative phosphorylation were significantly down-regulate d in suspension cells compared to adherent and had amongst the highest number of differentially expressed genes. pathways related to retinol, linoleate and nucleot ide metabolism were also significantly down-regulate d in suspension cell - lines. on the other hand, biosynthesis and metabolism of cholesterol were found to be most significantly up - regulated amongst metabolic pathways in suspension compared to adherent cells. in addition, pathways related to protein modification and fatty acid metabolism ( omega - 3 / 6 fatty acid metabolism, fatty acid desaturation, fatty acid biosynthesis and fatty acid elongation ) were up - regulated in suspension compared to adherent hek293 progeny cell lines. all results from the metabolic gene set analysis are provided in supplementary table s8. focusing on the pairwise comparisons between hek293 cell lines, 38 differentially expressed genes were identified common to all adherent to suspension pairwise comparisons ( red column in figs. 3b, 5a, supplementary table s5 ). three of the genes ( arrdc3, hmgcs1 and pcyox1l ) had the same directional gene copy number variation ( gain or loss ) compared to gene expression fold - changes ( up or down ) of all suspension compared to adherent cells, which may at least partly explain the differential expression of these genes between the groups. gene enrichment analysis of this set of 38 differentially expressed genes between adherent and suspension cells, performed using enrichr, predicted the cholesterol biosynthetic process pathway as the cellular pathway most affected by this expression variation ( fig. 5b ). this result further emphasizes the differential expression between adherent and suspension cells of genes involved in the cholesterol pathway, mentioned above. among the 38 common differentially expressed genes msmo1, idi1, npc1l1, insig1 and hmgcs1 are directly related to cholesterol biosynthesis ( go: 0006695 and / or go: 0008203, fig. 5b ). each of these genes had at least a two - fold increase in expression in the suspension cells compared with the adherent cell - lines. based on these findings, we sought to predict the effect of the differentially expressed genes between adherent and suspension hek293 cell lines on the cholesterol biosynthesis of the cells using ingenuity pathway analysis ( ipa ).