6 fold higher than in BMP2 6 h experiment. In addition to the two set Venn analyses, the overlap of genes regulated in all four sets of selleck inhibitor experiment and in three out of four sets was additionally analyzed. A summary of these genes is listed in Table 1, indicating their fold change in each treatment. Remarkably, only eight genes were sig nificantly altered after the treatment with BMP2 and TSA at both time points Gpr17, Lims2, Bcas1, Ptpre, Afap1l2, Dll3, G0s2, Gpd1. 65 genes were regulated in at least three out of four experimental sets. Most of these genes were regulated in the same direction when treated with BMP2 or TSA, and only a few genes exhib ited an opposed expression. Smad7 Papss2, Fam19a2, Cadps, Car8 and Efhd1 are examples for an opposed regulation of expression comparing BMP2 and TSA treated samples.

Smad7, Papss2, Fam19a2, and Cadps expression was suppressed after TSA treatment but induced after treatment with BMP2, whereas Car8 and Efhd1 expression was regulated in a reverse fashion. In accordance with the results from the two set Venn analysis, the number of co regulated genes was increased when the BMP2 24 h time point was included in the intersection analysis. However, among these genes, the expression of only a few was significantly stronger regulated after 24 h than after 6 h of both TSA and BMP2 treatment. Especially, the expression of Gpr17, Lims2, Bcas1, BMP4, Enpp6 and Gm98 was significantly reduced in 24 h compared to 6 h experiments. It should be mentioned that, among those genes regulated by BMP2 6 h and 24 h and TSA 24 h, several genes known to be involved in BMP2/4 sig naling, like Bmp4, Smad7, Fst and Bambi were detected.

We also performed hierarchical clustering of the mi croarray data using the clustering option of dChip to illustrate the overall relationship between regulated genes. All genes regulated in at least one of the analyzed conditions were included using the same stringent criteria as above. The clustering led to two major clusters, one including genes upregulated, the other including genes downregulated upon either treatment. Genes up regulated after each treatment were further divided into three sub clusters, grouping genes upregulated after treat ment with BMP2 or TSA alone or both BMP2 and TSA. Each sub cluster could be subdivided into smaller groups of genes that represent individual time points.

Within the cluster of downregulated genes, also three sub clusters could be distinguished, containing genes downregulated after treatment with BMP2 or TSA alone and downregulated after both Drug_discovery treatments. To investigate the specific biological functions of co regulated groups of genes, we used the DAVID Database for functional annotation of clustered genes. Functional annotation clustering allows the clas sification of regulated genes according to their functional relevance. Each of the six sub clusters obtained in the hierarchical clustering was independently annotated.