Site-directed mutagenesis was performed to mutate the SF-1 and NF-Y binding sites in the −398FSHβLuc reporter gene. A, Transient transfections of mutant FSHβLuc plasmids were performed in LβT2, αT3-1, and NIH3T3 cells. The mutant plasmids are depicted in the diagram at left. Results represent the mean ± sem of five independent experiments, each performed in triplicate (n = 15). The letter a denotes a statistically significant difference of the NF-Y mutant plasmid as compared with wild type in LβT2 cells (P < 0.05); the double letter aa denotes a statistically significant difference between the NF-Y and SF-1 double or triple mutants and the NF-Y single mutant in LβT2 cells (P < 0.05); the letter b denotes a statistically significant difference of the mutant plasmid as compared with wild type in NIH3T3 cells (P < 0.05). B, Follistatin responsiveness of −398FSHβLuc does not require the NF-Y and SF-1 binding sites. LβT2 cells were transiently transfected with mutant FSHβLuc plasmids and treated with either vehicle (control) or 100 ng/ml follistatin for 24 h (FS-treated, hatched bars). The mutant plasmids are depicted in the diagram at left. To facilitate comparisons between promoter responses, the control samples were set at 1 and the FS-treated samples are shown relative to this control. Results represent the mean ± sem of four independent experiments, each performed in triplicate (n = 12). Bars marked with φ are statistically significant from the untreated controls (P < 0.05).