FIG 6.

NGFIB and NURR1 possess the ability to enhance HSD3B1 promoter activity. (A) Luciferase reporter assays of NGFIB and NURR1 for the HSD3B1 promoter. Reporter constructs containing serial deletions of the HSD3B1 promoter (positions −1059 to +274, −599 to −1, −200 to −1, and −100 to −1) as well as the mutant derivative for the NBRE (Mut; positions −124 to −117) were transiently introduced (20 ng plasmid/well for each) into H295R cells with either an empty pEZ expression vector (500 ng) or an expression vector containing the coding sequence of human NGFIB or NURR1 (500 ng). After recovery for 24 h, cells were lysed, and luciferase activity was measured. Deletion constructs are numbered relative to the transcription initiation site. Shown are representative data from replicate experiments with similar results. Values represent the means ± SEMs (n = 4). *, P < 0.01, Bonferroni test. (B) Luciferase reporter assays of NGFIB and NURR1 for the isolated NBRE. A luciferase reporter construct (20 ng) that contains either nine copies of the NBRE sequence of HSD3B1 (wild type) or those of the mutated sequence was transiently introduced into H295R cells with increasing doses of the expression vector for NGFIB or NURR1 (50, 400 ng). Values are the means ± SEMs (n = 4). (C) Luciferase reporter assays examining the effect of coexpression of NGFIB and NURR1 on the promoter activities of HSD3B1. The reporter plasmid containing the HSD3B1 promoter (positions −599 to −1) (20 ng) was transiently introduced into H295R cells with the indicated combinations of expression plasmids for NGFIB and NURR1. +, 200 ng, ++, 400 ng. Values are the means ± SEMs (n = 4).