Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 Table 2 Selection of genes regulated by NGF withdrawal identified in this study GO pathway AP1/ATF family Cell death DNA damage-inducible ER stress Fatty acid/cholesterol metabolism FOXO target MAPK pathways Miscellaneous Myc network PI3K-Akt pathway Gene symbol CREM JunD Bmf Casp12 Casp3 Casp4 Cycs GADD45a ATF3 Ddit3 Eif2ak3 Herpud1 Myd116 Hmgcr Insig1 Sc4mol Txnip Dusp6 RhoQ Areg Bhlhb2 Btg1 Btg2 Chrna7 Cnskr3 Pkib Tpo1 Id2 Mxi1 Mycn Ndrg1 Ptma PIK3IP1 PIK3r1 Gene name cAMP responsive element modulator Jun D proto-onocogene Bcl2 modifying factor caspase 12 caspase 3, apoptosis related cysteine protease caspase 4, apoptosis related cysteine protease cytochrome c, somatic Growth arrest and DNA damage-inducible 45 alpha activating transcription factor 3 DNA damage-inducible transcript 3/CHOP10 eukaryotic translation initiation factor 2 alpha kinase 3/PERK homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 myeloid differentiation primary response gene 116 3-hydroxy-3-methylglutaryl-coenzyme A reductase insulin induced gene 1 sterol-C4-methyl oxidase-like thioredoxin interacting protein/Vdup1 dual specificity phosphatase 6/Mkp3 Ras homologue gene family, member Q amphiregulin basic helix-loop-helix domain containing, class B2 B-cell translocation gene 1, anti-proliferative B-cell translocation gene 2, anti-proliferative cholinergic receptor, nicotinic, alpha polypeptide 7 Cnksr family member 3 (Magi1) protein kinase (cAMP-dependent, catalytic) inhibitor beta developmentally regulated protein TPO1 Inhibitor of DNA binding 2 Max interacting protein 1 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived N-myc downstream regulated gene 1 similar to HGFL protein phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1/p85 Page 8 of 17 -NGF CEP v v +NGF +NGF 1.51 1.25 1.74 1.60 1.91 1.31 1.46 1.23 1.73 1.31 1.60 1.03 -1.88 -1.19 1.45 1.41 2.89 -1.69 4.11 1.37 2.28 1.54 2.17 1.17 1.81 -2.79 -4.69 -3.95 3.68 -6.68 2.51 -5.90 -2.35 1.78 3.63 2.16 3.14 2.39 2.41 -1.75 2.22 -2.89 3.18 -1.93 5.28 1.85 1.09 -1.76 -1.51 -1.36 2.46 -5.66 1.16 -5.50 -2.36 1.44 1.64 2.16 1.54 1.02 2.04 -1.31 1.77 -1.87 2.07 -1.37 4.56 1.90 The fold increase in RNA level after NGF withdrawal or after NGF withdrawal in the presence of CEP-11004 (average of 3 independent experiments) is shown for a selection of genes that have been grouped according to functional activity. The level of gene expression in the presence of NGF was set to 1. Negative fold changes refer to down-regulation. Only genes with a FDR-adjusted p-value of < 0.01 are shown. also validated by quantitative PCR: ndrg1 and mxi1. Both of these genes are associated with the Myc gene regulation network and are induced after NGF withdrawal by 3.18- fold and 2.22-fold respectively. Quantitative PCR con- firmed the increase in mRNA levels for both of these genes (Figure 5A-C). The protein levels of selected regulated genes increase after NGF withdrawal We examined the effect of NGF withdrawal on the levels of the proteins encoded by the 5 selected genes and their localisation. In immunoblotting experiments, we observed a significant increase in the levels of the Trib3 and Ddit3 proteins by 16 hours after NGF withdrawal. In contrast, when sympathetic neurons were deprived of NGF in the presence of 400 nM CEP-11004 for 16 hours, there was no significant increase in the levels of these proteins when compared to neurons cultured in the presence of NGF (Figure 6A). Levels of Trib3 and Ddit3 protein and their subcellular localisation were also studied by immunofluor- escence (Figure 6B). In the presence of NGF, Trib3 levels were low and levels of Ddit3 were almost undetectable.