Structure and activity of the human PDPN 5'-flanking region in MG-63 and Saos-2 cells. A) Nucleotide sequence and putative regulatory elements within bp -857/+205 of the 5'-flanking region of the human PDPN gene. The major transcription start site is indicated with an asterisk at +1. Putative regulatory elements highlighted by underlining were identified using the Transfac database. Shown are only sites which are conserved between the human, murine and rat promoter. Numbers on the left and right refer to the nucleotide position relative to the major transcription start site. The scheme below shows the relative positions of these putative transcription factor binding sites within bp -1885/+205. AP, activating enhancer binding protein; C/EBPβ, CCAAT/enhancer binding protein β; ETF, EGFR-specific transcription factor; ICSBP, interferon consensus sequence binding protein; NF-1, nuclear factor 1. B) Schematic representation of stepwise 5' deletions of the PDPN 5' flanking region cloned upstream of the luciferase reporter gene into the pGL3 vector. Negative numbers indicate the 5' end of the promoter fragment relative to the major transcription start site at +1. C) Luciferase activity of 5' deletion constructs were assessed in MG63 and Saos-2 cells 24 h after transfection with equal stoichiometric plasmid amounts and a constant amount of internal control Renilla luciferase plasmid as described under "Methods". Luciferase activity was normalized to Renilla activity and is relative to the longest bp -1885/+171 construct, which was set at 100%. The average ± S.D. for three to six independent transfection experiments, each performed in triplicates, is shown. Statistical analysis was performed using student's t-test, and is as follows: *, p < 0.05, **, p < 0.01, ***, p < 0.001. The statistical analysis indicates a significant promoter activity difference between the consecutive deletion-constructs.