Fig 2: Slit2 signaling stabilizes BAF complex subunits and stimulates EWS::FLI1 transcriptional output.(A) Slit2 silencing reduces the protein levels of BAF complex subunits. A673, TC32, ES1, and PDX1 Ewing sarcoma cells were transfected with Slit2 siRNAs or control siRNAs and the levels of indicated BAF subunits were examined by immunoblotting. (B) The cdc42 inhibitor ML141 restores the protein levels of BAF subunits in Slit2-silenced cells. Ewing sarcoma cells were transfected with Slit2 siRNAs or control siRNAs and were treated with or without 5 μM ML141. The levels of BAF subunits were assessed by immunoblotting. (C) Slit2 silencing reduces EWS::FLI1 target gene expression in Ewing sarcoma. A673, TC32, ES1, and PDX1 Ewing sarcoma cells were transfected with Slit2 siRNAs or control siRNAs and the RNA expression of indicated genes was examined by qRT-PCR and is presented after normalization to the levels in control siRNAs transfected cells (blue). The expression of EWS::FLI1 target genes is reduced in Slit2-silenced cells (red). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Ewing sarcoma depends on Slit2.(A) Slit2 silencing inhibits Ewing sarcoma proliferation. Slit2 was silenced by siRNAs in four different Ewing sarcoma cells and cell proliferation was assessed by the IncuCyte live-cell imaging system. Slit2 silencing was verified by immunoblotting (bottom). (B) Recombinant Slit2 rescues proliferation arrest induced by Slit2 silencing. Ewing sarcoma cells were transfected with Slit2 or control siRNAs and were treated with or without recombinant Slit2 (250 ng/ml). Cell proliferation was assessed by IncuCyte. (C) Slit2 silencing does not induce apoptosis or neuronal differentiation. Slit2 was silenced in A673 and PDX1 cells and the expression of the indicated protein was examined by immunoblotting. (D) Slit2 silencing does not induce senescence. Slit2 was silenced by siRNA (top) or by two different shRNAs (middle) in A673 cells and cells were stained for senescence-associated beta-galactosidase. MCF7 breast cancer cells were treated with 1 μM doxorubicin for 2 hours and 4 days later, stained for senescence-associated beta-galactosidase (bottom). Doxorubicin-treated MCF7 cells displayed roust senescence-associated beta-galactosidase activity as we reported previously [30] whereas Slit2-silenced A673 cells did not detectably display senescence-associated beta-galactosidase activity. Scale bars: 100 μm.

Fig 4: EWS::FLI1 induces Slit2 expression.(A) Reduced Slit2 protein levels in A673 cell secretome upon EWS::FLI1 silencing. The quantification is based on spectral counting by mass spectrometry. (B) EWS::FLI1 silencing in A673 cells results in reduced Slit2 expression levels. A673 cells were infected with lentiviruses expressing a shRNA against FLI1 C-terminal region or control shRNA and were selected with 2 μg/ml puromycin for 2 days. The EWS::FLI1, Slit2, and β-actin mRNA levels were examined by quantitative real-time RT-PCR (qRT-PCR; left). Whole cell lysates were prepared and immunoblotting was performed using antibodies against FLI1 C-terminus, Slit2, and tubulin (right). ***p < 0.001 compared with control shRNA-expressing cells. (C) EWS::FLI1 expression in human mesenchymal stem cells results in increased Slit2 expression levels. Human mesenchymal stem cells were infected with lentiviruses expressing EWS::FLI1 or empty vector and were selected with 2 μg/ml puromycin for 2 days. The EWS::FLI1, Slit2, and β-actin mRNA levels were examined by qRT-PCR (left). Whole cell lysates were prepared and immunoblotting was performed using antibodies against FLI1 C-terminus, Slit2, and tubulin (right). ***p < 0.001 compared with control shRNA-expressing cells.

Fig 5: Slit2 signaling stimulates the transformed phenotype of Ewing sarcoma cells.(A) shRNA-mediated silencing of Slit2. A673 and PDX1 cells were infected with lentiviruses expressing two different shRNAs against Slit2 or control shRNA and were selected with 2 μg/ml puromycin. Four days after infection, the silencing of Slit2 was verified by immunoblotting. (B) The silencing of Slit2 impairs anchorage-independent growth. Cells in (A) were plated in semi-solid medium. Two weeks after culture, colonies were counted and photographed. ***p < 0.001 Scale bars: 100 mm. (C) The silencing of Slit2 impairs sphere formation. Cells in (A) were plated in ultra-low attachment 6-well plates. Two weeks after culture, spheres were counted and photographed. ***p < 0.001 Scale bars: 100 mm.