Transcriptional activation of spleen-specific (A), testes-specific (B), and steroidogenic (C) promoters by WT or mutant SF1 expression vectors were assayed by transient cotransfection of the expression vectors using the Promega Dual Luciferase assay system. For SF-1 binding elements in the reporters, see Supplemental Table 3. (A) Transcriptional activation of the spleen development–specific TLX1 promoter by WT and mutant SF1 constructs was studied in COS-7 cells. Ve, empty vector control. The TLX1 promoter–luciferase reporter construct is shown below, with the TLX1 promoter, transcription start site (arrow), exon 1 harboring 2 SF-1 binding sequences (spheres), and the luciferase reporter gene (Luc). Numbering is relative to the TLX1 transcription start site, at position +1. (B) Activity of the SOX9 testis-specific TESCO-luciferase enhancer, harboring both SF-1 and SRY/SOX binding sites, was measured in COS-7 cells. Transfections were performed using empty vector control (–) or WT or mutant SF1 expression vectors, either alone or together with Sry-myc or Sox9 expression vectors (3). A schematic illustration of the reporter construct is shown below. (C) Activity of the steroidogenic CYP11A1 (left), CYP17A1 (middle), and HSD3B2 (right) promoter reporter constructs (27) transfected with the SF1 constructs into nonsteroidogenic HEK293 cells. Results represent mean ± SEM relative luciferase activity of 4–5 independent experiments performed in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT.