Figure 5.

EWS-FLI1–induced NOTCH pathway activation induces p53 and p21^WAF1/CIP1 in TC252 cells. A, immunoblot monitoring NOTCH3 processing, P^Ser15-p53 and p21 induction in the absence and presence of 10 μmol/L GSI in two ESFT cell lines (TC252 and WE68) transfected with either nontargeting shRNA or EF30. Note that GSI reduced overall p53 expression, whereas Ser15 phosphorylation was not affected. B, cotransfection of siRNA to JAG1 or of a NUMB expression vector with EF30 reverts p53 and p21^WAF1/CIP1 abundancy to control levels, whereas ectopic JAG1 and DLL1 induce p53 and p21 similar to EWS-FLI1 silencing. For negative control, transfection of a nontargeting shRNA (left) and of an empty expression vector (right) are shown. NICD3 transfection served as a positive control. C, regulation of JAG1 in response to EWS-FLI1 silencing is confined to a 570-bp genomic region upstream of the JAG1 transcription initiation site. Firefly luciferase reporter gene assays for a 0.57-kb JAG1 promoter fragment were performed in three ESFT cell lines (TC252, SK-N-MC, and WE68) transfected with EF30 or control (empty vector or nontargeting shRNA). Fold changes in the transcriptional activity of the JAG1 promoter sequences after EWS-FLI1 knockdown were calculated as follows:

$$\text{Fold}\text{change}=\frac{{[-574/-1-\text{JAG}1.\text{Luc}/\text{TKp}.\text{rhL}]}^{\text{EWS}-\text{FLI}1\text{shRNA}}/{[-574/-1-\text{JAG}1.\text{Luc}/\text{TKp}.\text{rhL}]}^{\text{Scrambled}\text{shRNA}}}{{[\text{TKp}.\text{Luc}/\text{TKp}.\text{rhL}]}^{\text{EWS-FLI1shRNA}}/{[\text{TKp}.\text{Luc}/\text{TKp}.\text{rhL}]}^{\text{Scrambled}\text{shRNA}}}$$

Mean values for fold change from three to five independent experiments, each performed in triplicate are shown. D, ectopic expression of JAG1 in the presence of either siRNA to HEY1 or of GSI reverts p53 abundancy almost to control levels.