Cells were seeded in 96 well plates and cultured under proliferating conditions and any other enquiries either tested during proliferation with or without hypoxia or were further used for differentiation studies. Subsequently differentiation was induced by withdrawal of the growth factors and cells were either incubated at 20% O2 or 3% O2 for an additional time period of 24 h and 72 h. The Wst 1 reagent was added to a final dilu tion of 1,10 for 2 h and the formazan produced by the metabolic activity of the cells was measured at a wave length of 450 nm using a plate reader. FACS analysis Cell cycle analysis For cell cycle analysis, proliferating or differentiating cells were harvested and fixed in ice cold 70% ethanol for 1 h at 20 C.

Prior to FACS measurement fixed cells were incubated with 1 mg ml RNase A for 30 min at 37 C following incuba tion with 50 ug ml propidium iodide for 30 min at 37 C. DNA content was measured by flow cytometry and analyzed by using the Cell Quest Pro software. Aggregated cells and debris revealed by for ward scattering were filtered out of the data set prior to analysis. To quantify G1, S, and G2 M populations, set tings for 2N and 4N peaks were defined within each experiment from the G1 S cells and applied to all sam ples within a given experiment. Antibody staining of neuronal proteins For the detection of bIII tubulin positive cells, cells were detached, centrifuged at 100g at room temperature, washed with PBS without Ca2 Mg2 and fixed with 1% PFA in PBS for 15 min. Then, cells were resuspended in washing buffer and stored at 4 C in the dark.

After centrifugation cells were resuspended in saponin buffer containing diluted mouse monoclonal FITC conjugated b III tubulin antibody and incubated for two hours at RT. Cells were washed twice with saponin buffer and resuspended in wash buffer for analysis. Mea surement was done using FACSCalibur in combination with Cell Quest Pro software. TUNEL assay and staining Apoptotic cells during differentiation were detected with an in situ cell detection kit. Detached cells were fixed with 1% PFA PBS for 15 min at RT. Afterwards cells were centrifuged and washing buffer was added. Until labelling, samples were stored at 4 C. For permeabiliza tion and labelling, samples were centrifuged and washed with PBS followed by an incubation with permeabiliza tion solution for two minutes on ice.

After an additional washing step with PBS, cells were incubated with TUNEL reaction mixture for Entinostat 1 h at 37 C at RT. As a positive control, cells were treated with DNase I for 10 minutes. As a negative control a sample treated with labelling solution was used. Subsequently cells were washed twice with PBS and a final volume of 250 ul PBS was added. The samples were measured and analysed with FACS Calibur and Cell Quest Pro Software. Western blot analysis For western blot analysis total cell extracts were pre pared.