Figure 4.

ETV6 mutants are deficient in transcriptional repression and act in a dominant-negative manner. (a) Schematic of the pGL3 reporter constructs harboring the MMP3 and PF4 promoters upstream of the firefly luciferase gene. Black rectangles represent core ETS DNA-binding motifs. (b) Protein blot analysis of ETV6 expression in HeLa whole-cell lysates. (c) HeLa cells were cotransfected with the pGL3-MMP3 reporter construct, a pHAGE expression vector (empty vector, wild-type ETV6 or mutant ETV6) and pCS2 Renilla luciferase. Firefly to Renilla luciferase ratios (Fluc/Rluc) were calculated to control for transfection efficiency. Bars show the mean (+ s.e.m.) fold change in the Fluc/Rluc ratio relative to empty vector. Data represent at least two individual experiments for each condition with duplicate measurements. Pairwise Student’s t tests were performed comparing each condition to wild type (**P < 0.0005). (d) Experiments are as in c except that the pGL3-PF4 reporter construct was used. Data represent at least three individual experiments for each condition with duplicate measurements. Pairwise Student’s t tests were performed comparing each condition to wild type (**P < 0.0005). (e) HeLa cells were cotransfected with 50 ng of wild-type ETV6 expression vector with increasing amounts (50, 150 and 250 ng) of ETV6 expression vector encoding the Pro214Leu, Arg369Gln, Arg399Cys or monomeric Arg399Cys mutant, pGL3-PF4 reporter construct and pCS2 Renilla luciferase. Bars show the mean (+ s.e.m.) fold change in the Fluc/Rluc ratio relative to empty vector. Data represent at least three individual experiments for each condition with duplicate measurements. Pairwise Student’s t tests were performed comparing each condition to wild type alone (*P < 0.005, **P < 0.0005; NS, not significant).