Transcriptional regulation of RANKL gene promoter. (A) Mapping of the domain of RANKL
GSK2334470 mediating transcription activity using transient transfection assay in SaOS-2 cells. Various constructs were generated by PCR amplification. The construct length (left) and its relative luciferase activity (right) are indicated. Luciferase activity was measured using a dual luciferase reporter system as described under Experimental procedures. Data were derived from a representative experiment in triplicate after normalization to Renilla luciferase activity (internal controls). Each bar is means± SD. (B) Sequences of the 72 bp (?172 to ?100 bp) of RANKL regulatory region. E2F consensus binding site was indicated in bold. (C) E2F1 regulated RANKL promoter activity. pGL2-RANKL1890 promoter luciferase construct was transfected into SaOS-2 cells. The promoter activity was examined in the presence and absence of E2F1
expression vector (E2F1 CMV). (D) pGL2-RANKL1890 promoter luciferase construct was transfected into SaOS-2 cells. The promoter activity was examined in the presence and absence of the shRNA against E2F1, E2F2, and E2F3, respectively. Luciferase activity was measured using a dual luciferase reporter system as described under Experimental procedures. Data were derived from a representative experiment in triplicate after normalization to Renilla luciferase activity (internal controls). Each bar is means ± SD.
