Figure 2. Robo3 serves as the NELL2 receptor in Ewing sarcoma.

(A) Robo3 RNA expression in Ewing sarcoma tumors and cell lines (n = 3).

(B) NELL2 ligand-binding assays in A-673 cells. NELL2-FLAG bound to control siRNA transfected cells, and the binding was abolished by Robo3 silencing.

(C) NELL2 ligand-binding assays in COS cells. NELL2 fused to alkaline phosphatase (NELL2-AP) or AP alone was produced from transfected 293T cells and was incubated with COS cells that were transfected with vector, Robo3, or Robo1. The binding of NELL2-AP or AP to cells was visualized by AP reaction. NELL2-AP specifically bound to Robo3-expressing COS cells. Scale bars: 60 μm.

(D) NELL2 silencing results in accumulation of lentivirally expressed Robo3-FLAG on A-673 cell surface, which was reversed by recombinant NELL2. The quantification of the fraction of cells with surface Robo3-FLAG staining, based on the counting of >200 cells, is shown on the right. *p < 0.05; scale bars: 10 μm.

(E) Robo3 silencing inhibits Ewing sarcoma proliferation. A-673, EW8, TC32, and TC71 cells were transfected with Robo3 siRNA pool or control siRNA pool, and cell proliferation was assessed by IncuCyte (top). Robo3 silencing was verified by immunoblotting (bottom).

(F) Robo3 siRNAs targeting different regions of Robo3 inhibit A-673 cell proliferation. A-673 cells were transfected with 6 Robo3 siRNAs that target different regions of Robo3. Cell proliferation was assessed by IncuCyte (top). Robo3 silencing was verified by immunoblotting (bottom).

(G) NELL2 requires Robo3 to simulate Ewing sarcoma proliferation. A-673 cells were transfected with NELL2 siRNAs, Robo3 siRNAs, and/or control siRNAs and were treated with or without recombinant NELL2 (250 ng/mL) as indicated. Cell proliferation was assessed by IncuCyte.

(H) Robo3 serves as the NELL2 receptor in Ewing sarcoma.