Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 Page 9 of 17 Figure 5 Validation of gene induction after NGF withdrawal by real time PCR. Rat sympathetic neurons were cultured in vitro for 6 days and then maintained for 16 hours in 1) medium containing NGF, or 2) medium without NGF supplemented with anti-NGF antibody or 3) medium without NGF containing anti-NGF antibody and the compound CEP-11004 at 400 nM. RNA was then isolated using an RNeasy kit. The experiment was performed in triplicate and the resulting RNA samples were then processed and an Affymetrix rat exon microarray analysis was carried out. A, The fold increase in RNA level after NGF withdrawal or after NGF withdrawal in the presence of CEP-11004 determined by exon array analysis (average of 3 independent experiments) is shown for 5 representative genes. For each gene, the level of expression in the presence of NGF was set as 1 and the expression levels for the other treatments were normalised relative to this. B-C, mRNA levels for the 5 genes were also measured by real time PCR using RNA prepared from three independent sets of neurons and normalised to the housekeeping genes gapdh (B) or hprt1 (C). The data shown represents the average of three independent experiments ± SEM. Statistical comparisons were made between +NGF and -NGF or -NGF and +CEP-11004 for each gene. *p < 0.01. Levels of these proteins started to increase after ~8 hours peaking at 12-16 hours after NGF withdrawal. Trib3 was localised in both the nucleus and cytoplasm, whereas Ddit3 was localised mainly in the nucleus after NGF with- drawal. However, in the presence of CEP-11004, the levels of both proteins were decreased significantly to almost basal levels and more importantly were not detected in the nucleus. The protein levels of the other three genes: Txnip, Ndrg1 and Mxi1 were also studied by immunoblot- ting and immunofluorescence (Figure 6). Significant but modest increases in the levels of the Txnip, Ndrg1 and Mxi1 proteins were seen after NGF withdrawal and CEP- 11004 reduced this to varying degrees. The increase in Txnip protein level after NGF withdrawal was smaller than that seen at the transcriptional level. The effect of CEP-11004 was also not as significant at the protein level. The increase in the level of the Txnip protein and its loca- lisation after NGF withdrawal were also studied by immu- nofluorescence. The Txnip protein was clearly seen at 8 hours after NGF withdrawal in both the nucleus and cyto- plasm and this was followed by a steady increase in pro- tein levels over time. Both of the Myc pathway-associated proteins, Ndrg1 and Mxi1, also increased in level after NGF withdrawal and CEP-11004 reduced this increase (Figure 6). The txnip and trib3 promoters contain potential c-Jun binding sites We previously showed that three of the genes that are induced after NGF withdrawal in sympathetic neurons, c- jun, dp5 and mkp1, are direct targets of c-Jun [24,26,34]. The induction of these genes after NGF deprivation is strongly reduced by CEP-11004 and the c-jun, dp5 and mkp1 promoters contain functionally important ATF sites (Figure 7A) that have been shown to bind c-Jun/ ATF2 heterodimers in chromatin immunoprecipitation assays and EMSA experiments [24,34]. Some of the induced genes identified in our exon array analysis might also be direct targets of c-Jun, in particular those whose mRNA induction after NGF withdrawal is strongly sup- pressed by CEP-11004, for example txnip and trib3 (Fig- ure 5B and 5C). We therefore searched for conserved potential c-Jun binding sites in the promoter, first exon and first intron of the rat txnip and trib3 genes (Figure 7B and 7C). The txnip promoter contains an ATF site (TGAGGTAA), 919 bp upstream of Exon 1 in the rat gene, that is identical in sequence to the reverse comple- ment of the jun2 TRE site in the c-jun promoter (Figure 7A and 7B). This site is conserved in the rat, human, and cow txnip genes and contains two base changes in the mouse gene (Figure 7B). In the case of trib3, we identified a conserved ATF site (TTACATCA) 14 bp upstream of Exon 1 in the rat gene (Figure 7C). This site is identical to the reverse complement of the ATF site in the dp5 promoter (Figure 7A) and is conserved in the rat, mouse and cow genes and only one nucleotide differs in the human trib3 gene (Figure 7C). The presence of these potential c-Jun/ATF2 binding sites in the promoters of the rat txnip and trib3 genes suggests that these genes might be direct targets of the MLK-JNK-c-Jun pathway. Discussion In 1988, it was first proposed by Martin et al. [12] that new RNA and protein synthesis is required for NGF