Supplementary Material regardinga Digital genomics Drosophila cDNA chip experiment

To examine changes in gene expression in stably transfected S2 cells after treatment with DMSO, cDNA microarray analysis was performed using total RNA from S2 cells at 3 h-post DMSO treatment with CuSO4 induction. Untreated-transfected S2 cells were used as a control. Isolation of total RNA was achieved using an RNeasy Mini Kit (Qiagen). cDNA microarray analysis was performed in duplicate using TwinChip Fly-6K, which contains a total of 6,112 genes (TwinChip™ is the trademark of the Digital Genomics high-density chip, which has two identical arrays on a slide). Labeling, hybridization, and washing of respective cDNAs were all performed according to the instructions provided by the manufacturer (Digital Genomics Co, Seoul, Korea; DMSO-untreated and-treated samples were labeled with cy(cyanin)-5 (Red) and with cy(cyanin)-3 (Green), respectively. Complete gene descriptions on the Digital Genomics high-density chip are given on the web site ( Fluorescent images of hybridized microarrays were obtained using image acquisition through a confocal laser scanning with a scanner (Packard BioChip Technologies). The expression ratio between treated and untreated control samples was determined, and quantification was based on a scanned image using GenePix 3.0 (Axon Instruments).

Out of 6,112 genes, 40 known or putative genes were identified with hybridization values greater than 1.5 (Table 1). Twenty nine genes were significantly up-regulated and 11 genes were significantly down-regulated. A gene encoding NOS was up-regulated 3.45 fold, whereas expression of theKr-h1 gene was increased 1.63 fold. Many other genes that were either up-regulated or down-regulated to values greater than 2.0 are involved in diverse cellular functions including gene/protein expression, cell signaling/communication, and cell metabolism. Functional characterization of these individual genes, as well as additional knowledge of the biochemical and genetic pathways, would be necessary for a complete understanding of the DMSO mechanism in S2 cells. Although we cannot exclude the possibility that other mechanisms are involved, we conclude that NOS and transcription factor Kr-h1 are important mediators for enhanced expression of recombinant human cyclooxygenase 1 in DMSO-treated S2 cells. These conclusions are based on our experimental results (Figures 2 and 3) of the paperand the cDNA chip data on the gene expressions of NOS and Kr-h1.

Table 1. Genes that were up- and down-regulated in DMSO-treated S2 cells.

Gene symbol / Fold / Putative identification / Functional
classification
Up-regulated genes
CG1317 / 7.08 / Unknown
CG12558 / 4.19 / Serine-type endopeptidase / CSC
Slpr / 3.78 / Protein serine/threonine/tyrosine kinase, JNK cascade / CSC
Nos / 3.45 / Nitric oxide synthase / CSC
CG1532 / 3.07 / Glyoxalase/fosfomycin resistance/dioxygenase / CM
Eip74EF / 2.97 / Ecdysone-induced protein 74EF / GPE
Tollo / 2.55 / Defense response, transmembrane signaling receptor / CSC
Mtp / 2.22 / Microsomal triacylglycerol transfer protein / CM
Toll / 2.10 / Transmembrane signaling receptor / CSC
Pros / 2.05 / Prospero, central nervous system development / GPE
Torso / 1.89 / Protein tyrosine kinase / CSC
DnaJ-1 / 1.84 / Chaperone, DnaJ-like-1 / COD
Pi3K92E / 1.83 / Phosphatidylinositol 3-kinase, lipid kinase / CSC
Pb / 1.83 / Proboscipedia, RNA polymerase II transcription factor / GPE
Trap 170 / 1.79 / Thyroid hormone receptor-associated protein / GPE
Gsb-n / 1.77 / Specific RNA polymerase II transcription factor / GPE
Chi / 1.75 / Transcription factor binding / GPE
RhoGAPp190 / 1.73 / Rho GTPase activator / CSC
Sgl / 1.72 / UDP-glucose 6-dehydrogenase / CM
Hsp27 / 1.71 / Heat shock protein 27 / COD
Df31 / 1.70 / Decondensation factor 31,chromatin organization / CGD
Rel / 1.69 / Specific RNA polymerase II transcription factor / GPE
Trio / 1.69 / Rho guanyl-nucleotide exchange factor / CSC
Pen / 1.66 / Protein transmembrane transporter / CSM
Nod / 1.66 / Kinesin motor (motor protein) / CSM
Scsalpha / 1.66 / Succinate-CoA ligase complex / CM
Kr-h1 / 1.63 / Transcription factor / GPE
Cas / 1.50 / Apoptosis inhibitor / CGD
Cyclin B / 1.50 / Cyclin-dependent protein / CGD
Down-regulated genes
CG1233 / 3.87 / Transcription factor / GPE
Tailup / 2.95 / Specific RNA polymerase II transcription factor / GPE
Ama / 2.58 / Amalgam, neural cell adhesion molecule I / CSC
GH02712 / 1.90 / Glucan 1,4-alpha-glucosidase / CM
CG6701 / 1.88 / DNA binding (H1, H5 family) / CGD
Cyp9b2 / 1.86 / Cytochrome P450 / COD
BicD / 1.86 / Presumed cytoskeletal element / CSM
Pms2 / 1.85 / DNA repair protein / COD
Brf / 1.76 / RNA polymerase III transcription factor / GPE
Pka-R1 / 1.62 / cAMP-dependent protein kinase R1 / CSC
Med / 1.59 / TGFbeta receptor signaling pathway / CSC

The putative identifications were assigned based on the FlyBase, and functional classifications were made as follows: CGD, cell growth/division; CM, cell metabolism; COD, cell organism/defense; CSC, cell signaling/communication; CSM, cell structure/motility; GPE, gene/protein expression.