Af ter TSA 6 h treatment, categories were enriched which contained genes with functions in histone modification, chromatin organization, transcription regulation and cell cycle control. Similar Ganetespib to the 24 h BMP2 treatment, the 24 h TSA treatment also showed a more diverse set of functional categories. Interest ingly, these categories resembled the categories enriched after BMP2 24 h treatment. This functional overlap is also reflected in functional annotation clustering of genes regulated in both BMP2 24 h and TSA 24 h sam ples. Clusters with genes involved in functions in plasma membrane, cell adhesion, cell communication, as well as genes involved in developmental processes were enriched.

This suggests that genes regulated after 6 h directly reflected the well established activity on gene regulation mediated by histone deacetylase inhibition, but that after 24 h already a secondary biological effect may have been observed. Validation of the microarray data with mRNA expression analysis For validation of the microarray data, we selected several genes and performed quantitative RT PCR. Gpr17, Bambi, Smad7 and Bmp4 were chosen from the lists of genes regulated in both TSA and BMP2 treatment. In order to obtain a more detailed view of the regulatory response, one additional time point and two additional TSA con centrations were used. All selected genes showed consistent expression patterns in RT PCR and the micro array experiments, although fold changes determined in the microarray analysis and the quantitative RT PCR dif fered significantly.

In addition to Bambi, Smad7 and Bmp4, known to be involved in BMP signaling, we de cided to analyze the expression of Bmp2 and the BMP target genes Id1 and Id2. While Bmp4 was downregulated upon TSA treatment, the expression of Bmp2 was sig nificantly upregulated in a concentration dependent manner after 6 h, but not after 12 and 24 h. Surprisingly, Id1 and Id2 expression was downregulated at 6 h, but increased after 12 h, resulting in a similar level of expression compared with BMP2 treated cells after 24 h, suggesting a partial BMP signaling independent effect on Id Brefeldin_A expression. We furthermore investigated Stat3, known to be an upstream regulator of BMP expression but also to co regulate astrocyte specific genes through the formation of a STAT3 p300 Smad complex. We also decided to analyze Wnt5a and Wisp1, both of which are involved in Wnt signal ing and are known to act upstream of BMP signaling. Stat3, as well as Wnt5a and Wisp1, were significantly upregulated upon TSA treatment in a time and concentration dependent manner.