Fig 2: EZH2 suppresses ERVs, LINE-1, and an interferon response downstream of NELL2 signaling.(A) Extracellular NELL2 signals to maintain histone H3K27me3 modification in ERVs and LINE-1 in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. Histone H3K27me3 modification in ERVs and LINE-1 was analyzed by chromatin immunoprecipitation. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (B) EZH2 expression is regulated by NELL2 signaling in Ewing sarcoma. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of EZH2 was analyzed by qRT-PCR. NELL2 silencing resulted in reduced EZH2 expression, which was restored by recombinant NELL2. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (C) The CD133low population displays low EZH2 expression, which was rescued by exogenous CD133. Cell populations in Figure 2 were analyzed for the expression of EZH2 by qRT-PCR. *p < 0.05 compared with the CD133high population and with the CD133low population expressing exogenous CD133. (D) NELL2 signaling maintains EZH2 binding to ERVs and LINE-1. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. The binding of EZH2 to ERVs and LINE-1 was assessed by chromatin immunoprecipitation. NELL2 silencing reduced EZH2 binding to ERVs and LINE-1, which was restored by recombinant NELL2. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (E) EZH2 suppresses an interferon response induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The expression of the indicated genes was analyzed by qRT-PCR. *p < 0.05 compared with cells transfected with control siRNA and empty vector and with cells transfected with EZH2. (F) EZH2 suppresses growth arrest induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. Cell proliferation was assessed using the IncuCyte live-cell imaging system. (G) EZH2 expression does not affect siRNA-mediated NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The protein levels of NELL2, IFNB1, Mx1, OAS1, and p21 were assessed by immunoblotting. Tubulin serves as a loading control.

Fig 3: Suppression of NELL2 signaling induces an interferon response in Ewing sarcoma.(A–E) siRNA-mediated silencing of NELL2 induces an interferon response in Ewing sarcoma cells. Ewing sarcoma cells were transfected with NELL2 siRNA pool or control siRNA pool and the expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by quantitative RT-PCR (left) and immunoblotting (right). *p < 0.05 compared with control siRNA transfected cells. (A) A673 cells, (B) EW8 cells, (C) TC32 cells, (D) TC71 cells, and (E) SK-N-MC cells. (F) shRNA-mediated silencing of NELL2 induces an interferon response in A673 cells. NELL2 was silenced by lentiviral expression of shRNA in A673 cells and the expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by qRT-PCR. *p < 0.05 compared with control shRNA expressing cells. (G) Extracellular NELL2 signals to suppress an interferon response in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by qRT-PCR. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. **p < 0.05 compared with cells transfected with NELL2 siRNA and left untreated or treated with recombinant NELL2.