Myoung-Sool Do, et al. Forward primer Reverse primer Table 1. Primer Sequences for Real-Time PCR Candidate genes RGS2: Regulator of g-protein signaling 2 5'-CAA CTG CCC AGA AAA GGG TA-3' 5'-ATG GCA GGT CAC AGT CCT TC-3' TNFSF13B: Tumor necrosis factor (ligand) superfamily, member 13b 5'-ATG CAG AAA GGC AGA AAG GA-3' 5'-AAG GCA AGA AGT AAG GCG TGA-3' CH25H: Cholesterol 25-hydroxylase 5'-TGG CAA CGC AGT ATA TGA GC-3' 5'-AGG GAA GTT GTA GCC GGA GT-3' Eotaxin-1: A potent eosinophil chemoattractant 5'-AGA AAC CAC CAC CTC TCA CGC-3' 5'-CAC AGC TTT CTG GGG ACA TT-3' MCP-1: Monocyte chemoattractant protein-1 5'-GCC TCC AGC ATG AAA GTC TC-3' 5'-CAG ATC TCC TTG GCC ACA ATG-3' VCAM1: Vascular cell adhesion molecule 1 5'-TCC GTC TCA TTG ACT TGC AGC ACC-3' isoform a precursor 5'-CAC CTG GAT TCC TTT TTC CA-3' IL1F5: Interleukin 1 family, member 5 5'-GCG GGG AAG ACT ATG TAA A-3' 5'-AAG GTT TCA GCA GAG CCG TTA-3' ADAMTS1: A disintegrin and metallo-protease with 5'-CTT TGC GCT GGA AGA AAA AC-3' thrombospondin motifs-1 preproprotein 5'-ACG GAG CGA GGG AGC TTT AGT T-3' FN1: Fibronectin 1 isoform 1 preproprotein 5'-CCA ACC TAC GGA TGA CTC GTG CT-3' 5'-GCT CAT CAT CTG GCC ATT TT-3' MMP15: Matrix metalloproteinase 15 preproprotein 5'-CGT GTC CTG CTT TAC TGC AAG-3' 5'-AGT CGG GGA AAC AGA AAC AAA-3' PTGES: Prostaglandin E synthase 5'-CAT GTG AGT CCC TGT GAT GGC-3' 5'-CTG CAG CAA AGA CAT CCA AA-3' GAPDH: Glyceraldehydes-3-phosphate dehydrogenase 5'-GAG TCA ACG GAT TTG GTC GT-3' 5'-TGA CAA GCT TCC CGT TCT CAG-3' replaced the medium of adipocytes on Day 18 with cortisol-free medium and incubated the culture with TNF-α for 24 h on Day 19. The cells were harvested on Day 20, and the gene expres- sion profiles were analyzed using the inflam- matory human arrays. From the 136 inflamma- tory genes present on the arrays, a total of 42 genes were expressed at either day 20 or day 20/ TNF-α (Fig. 2). Of these, 11 genes were two- fold up- or down-regulated. In TNF-α-treated SGBS adipocytes, 6 genes were up-regulated, and 5 genes were down-regulated (Table 2). The up- regulated genes were: regulator of g-protein sig- naling 2 (RGS2), Tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B), cholesterol 25-hydroxylase (CH25H), eotaxin-1, MCP-1, and vascular cell adhesion molecule 1 isoform a pre- cursor (VCAM1). The down-regulated genes were: IL-1 family member 5 (IL1F5), a disintegrin and metalloprotease with thrombospondin motifs-1 Yonsei Med J Vol. 47, No. 5, 2006 preproprotein (ADAMTS1), fibronectin 1 isoform 1 preprotein (FN1), matrix metalloproteinase 15 preprotein (MMP15), and prostaglandin e syn- thase (PTGES). Real-time PCR analysis of up- and down- regulated genes of TNF-α-treated SGBS cells The expression profiles found in the microarray experiments of TNF-α-treated SGBS cells were re- examined by real-time PCR with custom designed primers for candidate genes (Table 1). Of the 6 genes shown through array analysis to be up-regu- lated, only 3 genes, MCP-1, eotaxin-1, and VCAM1, were also shown by real-time PCR to be up-regu- lated. Of the 5 genes revealed by array analysis to be down-regulated, 4 genes, IL1F5, MMP15, FN1, and ADAMTS1, were found also by real-time PCR to be down-regulated. PTGES was not down-regu- lated according to either method (Table 3).