FIGURE 2.

EWS-FLI1 expression in COS7 cells leads to an increase in endogenous GLI1 protein expression and transcriptional activity. A, COS7 cells were transfected with EWS-FLI1 or an empty vector control. Whole cell lysates were subjected to SDS-PAGE and subsequent immunoblotting with GLI1, FLI1, or actin antibody. FLI1 immunoblotting confirmed the expression of EWS-FLI1 and actin was used as a loading control. The values below the top panel are the densitometry values and are given as fold increase over the empty vector control. B, COS7 cells were cotransfected with and without EWS-FLI1 and a pGL38xGli responsive luciferase construct. Twenty-four hours after transfection, the cells were treated with the cyclopamine or 75503 GLI inhibitor at a concentration of 30 μm in 1% DMSO for 24 h. The columns represent the means of the relative luciferase activity, which is calculated by dividing the luciferase activity by the Renilla activity used as a transfection control. The error bars are the standard deviations (*, p < 0.05; ***, p < 0.001 using a two-tailed Student's t test). Transfection assays were performed in triplicate and repeated three times. One representative experiment is shown in this figure. IB, immunoblotting.