Laboratories Of Cancer Pathology [T-K. Y.] and Epidemiology lS-Y. Korea Cancer Center Hospital. 215-4 Gongneung-Dong, Nowon-ku. 139-240. Korea.
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Figure 5. ARE mediates the transcriptional regulation of the GPX2 gene. A ; Comparison of the first exon and the 5 upstream regulatory regions of the human, mouse, and rat GPX2 genes. The short ARE core, long ARE consensus, GPX2ARE, ARE from the human HO-1 gene StREb ; , human NQO1, and human GCLm EpRE ; are aligned. The vertical bar indicates nucleotides that are identical between the human, mouse, and rat ARE as well as long ARE, HO-1 ARE. The abbreviations follow the standard IUPAC nomenclature M A or C, Deletion analysis of GPX2 promoter by transient transfection in A549 cells. A 2.569-kb portion of the GPX2 promoter was cloned into pGL3 basic luciferase reporter vector 2, 030 539 ; to monitor the activity of this promoter. Two deletion constructs 1, 029 539 and 140 539 ; were prepared by PCR. All three constructs were transfected into A549 cells transiently transfected with SS, Nrf2, or Keap1 siRNA, and luciferase activities were measured after 48 h. The three constructs had similar reporter activity. The 140 539 deletion construct displayed the maximum reporter activity. * Significant when compared with vector control transfected with respective siRNA; significant when compared with SS siRNA; significant when compared with Nrf2 siRNA. C ; Reporter constructs ARE-Mu1 and ARE-Mu2 ; bearing mutations in the ARE 140 539 ; were generated by using the 140 539 deletion construct. These constructs were transiently transfected into A549 cells already transfected with siRNA, and luciferase reporter activity was monitored. Mutation of the ARE core sequence completely abolished the reporter activity of the deletion construct. * Significant when compared with ARE-WT. D ; 201 bp 140 61 ; ARE enhancer sequence was amplified from the full-length promoter and cloned in pTAL vector. Mutations were introduced in the ARE core sequence ARE-Mu1 and AREMu2 ; by site-directed mutagenesis. These constructs were transfected into A549 cell lines transiently transfected with siRNA and luciferase activity was measured. Luciferase activities were normalized by measuring the Renilla luciferase activity from a cotransfected reporter vector. * Significant when compared with ARE-WT n 3 independent experiments ; . Values are mean SE from three different experiments P 0.05. 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