In an effort to identify specific alterations of gene transcription as a result of c-secretase activity, the transcriptomes of two CHO cell lines (biological triplicates were used in each case) with To identify genes whose transcription is affected by c-secretase activity, two starkly contrasting conditions were analyzed by cDNA microarray: genetically engineered enhanced c-secretase (left panel) and pharmacologically inhibited c-secretase (right panel) in CHO cell lines. For a schematic depiction of the strategy, the Notch-1 receptor signaling pathway is used as an example. After processing by the Furin protease and when activated by binding to its ligands Notch-1 is cleaved at the S2 position by the TACE protease, generating a substrate for c-secretase (1, 7) . Under enhanced (left panel) or inhibited (right panel) c-secretase activity, the cleavage of the substrate controls the release of the Notch intracellular domain (NICD) (2, 8) . With enhanced c-secretase, increased numbers of NICDs enter the nucleus and interact with CSL (3), leading to the transcription of target genes like Hes1 and Hey (4). The Hes1 transcription repressor inhibits transcription of target genes like NC3C1 (5), with the final consequence being reduced production of NC3C1 mRNA (6) . Thus, enhancing c-secretase leads simultaneously to gene-dependent increase (in the case of Hes/Hey) or decrease (in the case of NC3C1) of mRNA copy numbers. With inhibited c-secretase, reduced numbers of NICDs (9) lead to the transcription of less Hes1/Hey (10), to reduced inhibition of target genes like NC3C1 (11) and consequently to increased production of NC3C1 mRNA (12) . Inhibiting c-secretase thus leads to gene-dependent decrease (in the case of Hes/Hey genes) or increase (in the case of NC3C1) of mRNA copy numbers. Following mouse cDNA microarray analysis of both transcriptomes, top scoring candidates were evaluated and validated by real time PCR and further analyzed for changes of transcript levels between healthy and AD human brain cortices. doi:10.1371/journal.pone.0006952.g001 enhanced and inhibited c-secretase activity were analyzed and compared (strategy depicted in Fig. 1 -exemplified by Notch processing). The S-1 cell line overexpresses the four components of c-secretase (NCT, Aph1a, PS1 and Pen2) and was characterized by a marked increase in the level of PS1 heterodimers and an associated 8-fold increase in c-secretase activity compared to untransfected controls [17] . The other cell line consisted of the original parental wild type CHO cells incubated with DAPT, a well-known c-secretase inhibitor. We strategically chose those two conditions, overexpression of c-secretase and inhibition of its activity, to amplify the activity-dependent effects on gene transcription levels (i.e., amplification of the signal from the cDNA microarray). To reduce potential effects due to changes in the protein levels of the c-secretase subunits as opposed to changes in its activity that we are interested in, we used chemical inhibition (DAPT) of c-secretase activity instead of gene silencing, which ultimately leads to changes in protein levels [18] . Biological functions have indeed been reported mainly for the c-secretase subunit PS1, independently to the c-secretase activity. However, because treatment of CHO cells with DAPT has been recently reported to exacerbate the secretion of exosomes [19] , we cannot exclude at this stage that some detected genes may be exosomerelated in response to the DAPT treatment. The gene transcription levels of the two cell lines were analyzed using a mouse cDNA microarray ( Fig. 1 and Material and Methods) [20] because of the absence of a readily available DNA microarray based upon hamster gene sequences or cDNA clones. This lack has been noticed, and cross-species reactivity of a mouse microarray hybridized with CHO-derived samples has been investigated recently by De Leon Gatti et al. [21] . This group generated an EST-based CHO microarray and compared it with results from a mouse microarray and vice versa. They state that cross-species hybridization yielded 89.6% overlap in their arrays, noncontradicting results and led only to a decrease in sensitivity resulting in detection of fewer differentially expressed genes. Accordingly, we probably have not detected all differentially expressed genes, but we have detected a significant amount, including clusters of functional relevance. For example, Neprilysin, an Ab-degrading enzyme of functional relevance to AD that has been previously shown to be transcriptionally downregulated in PSEN1/PSEN2 double knock out fibroblasts and to exhibit reduced activity under chemical (DAPT) inhibition of c-secretase in mouse neurons [22] , was not detected in the current study. Collectively, this supports the use of CHO cells with a mouse microarray. The microarray data set discussed in this publication has been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and is accessible through GEO Series accession number GSE16379.