Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 Page 3 of 17 Figure 1 Timecourse of NGF withdrawal-induced apoptosis in sympathetic neurons. Rat sympathetic neurons were cultured in the presence of NGF for 6 days in vitro and then maintained in medium lacking NGF and containing anti-NGF antibody for the times indicated. Timelapse microscopy shows that sympathetic neurons develop an apoptotic morphology during the first 24 hours following NGF withdrawal with membrane blebbing (white arrows) and cell shrinkage occurring. Immunocytochemistry using a MAP2 antibody shows that between 4 and 8 hours after NGF withdrawal, neurites become fragmented and by 24 hours there are significantly fewer neurites compared to the control (0 hours). These morphological changes are accompanied by nuclear changes indicative of apoptosis visualised by Hoechst and TUNEL staining and these are seen as early as 2-4 hours after NGF withdrawal. Importantly, c-Jun becomes phosphorylated and is localised exclusively in the nucleus after NGF-withdrawal. Cytochrome c is released from the mitochondria and decreases in level. Finally, caspase-3 becomes activated and is clearly detected from 8 hours following NGF deprivation. The scale bar represents 20 μm. Gene expression profiling in sympathetic neurons after NGF withdrawal To identify new genes that may play a role in NGF withdrawal-induced apoptosis, we performed a gene microarray analysis using Affymetrix Exon arrays and RNA isolated from sympathetic neurons that had been cultured for 16 hours in the presence of NGF (+NGF), absence of NGF (-NGF) or absence of NGF but with the MLK inhibitor CEP-11004 (-NGF+CEP-11004) added to the medium (Figure 2). MLKs are upstream activators of the JNK pathway in sympathetic neurons and CEP- 11004 therefore blocks the increase in JNK activity and c-Jun phosphorylation and protects against NGF with- drawal-induced death. Three independent experiments were performed. Quality control and data analysis revealed good normalisation and reproducibility. An FDR-corrected p-value of 0.05 was used as an initial cut off to identify statistically significant differences in gene