Whereas the protein levels of GFAP was rather unchanged 24 h after treatment with TSA and BMP2, a strong increase of GFAP could be detected 7 days after treatment, indicating U0126 that the treatment with TSA and BMP2 led to an increase in astrogliogenesis dur ing differentiation of neurosphere cultures. The oligo dendrocyte markers Plp and Mbp were less clearly regulated on the protein level at both time points, but a small decrease of both markers could be detected 7 days after treatment. Microarray analysis of differentiating neurosphere cultures RNA samples and protein lysates were prepared 6, 12, and 24 h after treatment. We performed gene expression pro filing from cells treated for 6 h and 24 h using Affymetrix GenChip 420 2. 0. The raw data was analyzed using dChip software.

Genes were consid ered to be significantly regulated if their expression had changed more than two fold and had exceeded a minimal absolute difference of 100 comparing treated and mock treated cells with a confidence greater than 90%. Using these conditions 220 genes exhibited a differential expres sion in BMP2 treated cells after 6 h, and 573 genes were differentially regulated after 24 h. TSA treat ment led to 917 differentially expressed genes after 6 h and 982 after 24 h treatment. The top 25 genes regulated after TSA or BMP2 treatment are listed in the Additional file 1 Table S1 S4. To identify an overlap of regulated genes within TSA and BMP2 treatments, two set Venn analyses were performed, intersecting TSA 6 h, TSA 24 h, BMP2 6 h and BMP2 24 h experimental sets, respectively.

The inter section between two experimental sets is shown in individual Venn diagrams. The numbers of regulated genes for each treatment condition are depicted in the diagrams, while individual genes within the inter section are Dacomitinib listed in the Additional file 1 Table S5 S10. Comparing these two set Venn diagrams, it could be observed that the majority of regulated genes was unique for one treatment. only a smaller number of genes was located within the intersections between the two experi mental sets. The largest intersection of regulated genes was detected between TSA 6 h and TSA 24 h. The intersection between the BMP2 6 h and 24 h experi ments was marginally smaller. however more than half of the genes regulated after 6 h overlap with genes regulated after 24 h. Comparing the Venn analyses of TSA and BMP2 treated samples at the two different time points an increased number of co regulated genes could be detected from 6 h to 24 h. Whereas only 27 genes were regulated in both BMP2 6 h and TSA 6 h, the number of regulated genes in BMP2 24 h and TSA 24 h experimental sets increased 6 fold.