Expression Profile Of Human Lymphatic Endothelial Cells Cultured On Stiff (25 Kpa) Or Soft (0.2 Kpa) Matrix Conditions In The Presence Or Absence Of Gata2

Global transcriptome analysis showed that human lymphatic endothelial cells (LECs) grown on a soft matrix exhibit increased GATA2 expression, concomitant with a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including the key lymphangiogenic growth factor receptor VEGFR3.<br> Affymetrix GeneChip analysis revealed regulation of 2771 transcripts above or below a 1.4-fold change (log2 fold change &gt;0.5 or &lt;-0.5) threshold on soft versus stiff matrices. Moreover, 406 (27%) of the 1485 transcripts that were increased and 207 (16 %) of the 1286 transcripts that were decreased on soft matrix were regulated in a GATA2 dependent manner. Overall design: In total 18 samples were analyzed (first biological replicate is uploaded here). To identify differentially expressed genes between the ctrl stiff and ctrl soft groups, a stepwise analysis with 6 biological replicates was performed. First, exon set ID’s with an average expression lower than 5 were considered as not significantly expressed and excluded from the analysis. A threshold of 40% increase (&gt;0.5 log2 fold change) or decrease (&lt;-0.5 log2 fold change) of gene expression on the soft matrix (vs. stiff matrix) was considered for further analysis. For all genes with 3 or more exon probe set ID’s regulated above the defined thresholds, the average log2 fold change of the regulated exon probe ID’s was calculated and used to generate the final list of genes regulated by matrix stiffness. To determine which of the genes are regulated in a GATA2 dependent manner, a stepwise analysis with 2 biological replicates ctrl siRNA soft vs GATA2 siRNA soft groups was performed. The previously identified exon probe ID’s for genes differentially regulated between the ctrl stiff and ctrl soft groups were extracted and their average log2 fold change was calculated for the new data set. A threshold of 40% increase (&gt;0.5 log2 fold change) or decrease (&lt;-0.5 log2 fold change) of gene expression in the absence of the GATA2 was used to generate the final list of genes.