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Figure 1 Northern blot analysis of SERPINI1 and PDCD10 in various tissues. Human MTE multiple tissue expression array II (BD Clontech) was hybridized with 32P-labeled probe against A) SERPINI1, B) PDCD10, and C) control ubiquitin, respectively. Panel D shows the individual designations of all tissues examined.
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Figure 2 Relative expression levels of SERPINI1 and PDCD10 between normal and tumorous tissues. A) Semi-quantitative RT-PCR analysis of SERPINI1 and PDCD10 transcripts. RNAs used in this study were obtained from the normal human brain (Lanes 2, 6 & 10), human neuroglioma H4 cells (Lanes 3, 7 & 11) and human glioblastoma U-87 MG cells (Lanes 4, 8 & 12). M indicates the DNA size markers; and Lanes 1, 5 & 9 are the negative controls without templates in the PCR reactions. B & C) Northern hybridization analysis of PDCD10 (B) and ubiquitin (C) in paired normal/tumor tissue samples (BD Clontech). The array order of all tissues examined is shown in panel D.
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Figure 3 Analysis of the intergenic distance between PDCD10 and SERPINI1 and its promoter activity in various cell lines. A) The intergenic distance between PDCD10 and SERPINI1 was 851 bp, as determined from the results of 5'-RACE. The nucleotides between the two closest transcriptional start sites of PDCD10 and SERPINI1 are numbered from the centromeric (cen) end to the telomeric (tel) end. B) The promoter activity of the intergenic 851-bp fragment in either direction (toward PDCD10 or SERPINI1) was determined by the firefly luciferase assay in 6 different cell lines including human neuroglioma H4, human brain glioblastoma U-87 MG, human ovarian adenocarcinoma OVTW-59-4, human lung adenocarcinoma CL1-5, human cervical adenocarcinoma HeLa, and rat adrenal gland pheochromocytoma PC-12 cells.
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Figure 4 Sequence analysis of the intergenic region of the divergent PDCD10-SERPINI1 gene pair. Dot matrix alignment of the intergenic sequence of the human PDCD10-SERPINI1 pair with those of mouse (A) and rat (B). The units in both vertical and horizontal axis are the position numbers of the nucleotides in the intergenic region as shown in Fig. 3A. C) The complete nucleotide sequence of the intergenic spacer between human PDCD10 and SERPINI1. The putative transcription factor binding sites are underlined. The transcriptional start sites confirmed by 5'-RACE are indicated by bended arrows. The shaded nucleotides indicate the transcriptional start sites of the reference sequence reported by the NCBI database. The arrowheads indicate the cutting sites for the deletion assays described in Fig. 5.
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Figure 5 Identification of the minimal bidirectional promoter region of the PDCD10-SERPINI1 gene pair. The 5'- and 3'-deleted putative promoter fragments were individually cloned into the upstream of the firefly luciferase gene (LUC) in the pGL3-Basic vector followed by transient transfection into human neuroglioma H4 cells. The pRL-TK plasmid carrying Renilla luciferase gene was co-transfected as the internal control. The relative firefly luciferase activities were normalized to the Renilla luciferase activities to correct the transfection efficiency. The length and the position of each promoter insert are shown in scale. The numbers denote the positions of nucleotides with respect to +1, the first intergenic nucleotide next to the transcriptional start site of PDCD10, and +851, the intergenic nucleotide right next to the transcriptional start site of SERPINI1. The empty boxes represent inserted intergenic sequences. The shaded boxes represent the SV40 promoter.
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Figure 6 Identification of the enhancer and the repressive element within the intergenic region of the PDCD10-SERPINI1 gene pair. Various intergenic fragments were cloned upstream of either the SV40 or CMV promoter and of a luciferase reporter gene in the pGL3 vector. All constructs were transiently transfected into human neuroglioma H4 cells and the luciferase activity was measured. The resulting promoter activity was calculated relative to the control SV40 or CMV promoter activity which is artificially set at 100. A) The potential regulatory sequences from nt 176 to 851 of the intergenic region were cloned upstream of the SV40 promoter and their effects on the activity of the SV40 promoter were examined. B) Three intergenic fragments (nt 851-711, nt 710-474 and nt 473-176) in the PDCD10 direction were individually cloned upstream of the SV40 promoter to assess their effects on the activity of the SV40 promoter. C) Three different intergenic fragments (nt 711-851, nt 474-710 and nt 176-473) in the SERPINI1 direction were individually cloned upstream of the CMV promoter in a modified pGL3-Promoter vector. Their effects on the activity of the CMV promoter were then investigated. The numbering system for nucleotide position is the same as those described in Fig. 5. The length and the position of each promoter insert are shown in scale. In the names of constructs, S denotes the SV40 promoter and C denotes the CMV promoter.
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