Analysis of HNF1α promoter mutations. A, activation of mutant HNF1α promoter constructs. Normal (upper left panel), mutant HNF4α-binding site (upper right panel), mutant NKX6.1-binding site (lower left panel), and double mutant (lower right panel) HNF1α promoter constructs (1 μg) were cotransfected into 3T3 cells with NKX6.1 (1 μg) or HNF4α (1 μg) expression plasmid as indicated. Closed boxes indicate mutation, and open boxes indicate normal sequence. pcDNA3 was used as a control, and the relative value obtained was arbitrarily set to 1.0. B, luciferase (Luc) activity of the mutant NKX6.1-binding site HNF1α promoter in beta cells. βTC3 cells were transfected with 1 μg of the indicated normal (open box) and mutant NKX6.1-binding site (closed box) HNF1α promoter constructs. The luciferase value of the normal HNF1α promoter construct was arbitrarily set to 1.0. All samples were measured 24 h following transfection, and all experiments were repeated independently at least three times. ***, p < 0.001.