Figure 5

P1.1-mediated gene expression is positively correlated with USF binding to the E-box (-340). A) Transient cotransfection assays in Jeg3 cells of the P1.1-luciferase reporter gene plasmid in the presence of the indicated amounts of the dominant-negative A-USF construct. The luciferase activities were measured 24 h after transfection. To normalise measurements, the results from analogous cotransfection experiments with the empty expression plasmid were arbitrarily set to 1. Mean values ± S.E.M. of n = 3 experiments are shown. Differences between A-USF transfected cells and cells transfected with the empty expression vector were significant, as indicated by asterisks (* p < 0.05; ** p < 0,001). B) representative EMSA experiments with the labelled E-box probe and Jeg3 cell extracts left from reporter gene analyses. Jeg3 cells were transiently transfected with the P1.1-luciferase reporter gene plasmid plus 5 ng of the empty expression vector (- A-USF) or 5 ng of the A-USF expression plasmid (+ A-USF). In the presence of A-USF, binding of USF to the E-box probe is impaired. C) Scatter plot of luciferase activity vs. USF binding. Data points represent reporter gene and USF binding activities of individual extracts from reporter gene experiments with different amounts of A-USF- or empty expression plasmids. USF binding was quantified by applying the Image Quant software to EMSA experiments similar to the one shown in panel B. Data were statistically analysed with the Pearson product moment test. The correlation coefficient, r, and the p value are indicated.