Experimental validation of the putative promoters by reporter assays. (A) A schematic of reporter assay used to determine whether the identified putative promoter fragment can support transcription. Each putative promoter fragment was segmented into 750-bp fragments by PCR and cloned into the luciferase reporter construct, pGL3, in either forward or reverse orientations. The resulting reporter constructs were individually transfected into HT1080 cells and the resulting luciferase activity was measured. (B) Reporter activities of 17 putative promoter fragments are shown (remaining five of 22 were not tested). Relative reporter activity was determined by comparing the luciferase activity of the test fragment (Supplemental Table S3C) and the control genomic DNA fragments (Supplemental Table S3D). Promoter fragments with a significant reporter activity (exceeding three times the standard deviation of all control fragments) are highlighted in gray.
