D4 ES cell line, feeder-dependent, derived as described (Hogan BL et al. 1994. Manipulating the mouse embryo. A laboratory manual, second edition. Cold Spring Harbor Laboratory Press, pp255-272), was grown in DMEM, high glucose supplemented with 0.1 mM beta mercaptoethanol, 10% FCS, 400 ug/ml gentamycin and 500 pM Leukaemia Inhibitory Factor (LIF). For microarray experiments, ES cells, cultured twice without feeders, were diluted at 10 000 cells/ml in ES cell medium without LIF in the presence of 10 uM PD169316. Cell medium was changed every day and cells were lysed after 3 days. Total RNA from adherent cells were extracted using Qiagen RNeasy kit and treated with DNAse. Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 1999, Affymetrix). Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45oC on murine MG_U74A GeneChips. GeneChips were washed and stained in the Affymetrix Fluidics Station 400 and further scanned using the Hewlett-Packard GeneArray Scanner G2500A. The image data were analysed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of the array was arbitrarily set to 300.
