Fig 1: Slit2 signaling inhibits cdc42 in Ewing sarcoma.(A) Slit2 silencing activates cdc42 and Rac in Ewing sarcoma. A673 and PDX1 cells were transfected with Slit2 siRNAs or control siRNAs. Two days after transfection, the levels of GTP-bound, active cdc42 and Rac were examined by GST-PAK1 pull-down of whole cell lysate followed by anti-cdc42 and Rac immunoblotting. (B) Slit2 silencing barely affects active Rho A levels in Ewing sarcoma. A673 cells were transfected with Slit2 siRNAs or control siRNAs. Two days after transfection, the levels of GTP-bound, active Rho A were examined GST-Rhotekin-RBD pull-down of whole cell lysate followed by anti-Rho A immunoblotting. (C) Growth arrest induced by Slit2 silencing can be rescued by a cdc42 inhibitor, ML141. A673 and PDX1 cells were transfected with Slit2 siRNAs or control siRNAs and were treated with or without 5 μM ML141. Cell proliferation was assessed by IncuCyte. (D) Both Slit2 and NELL2 are necessary to suppress cdc42 in Ewing sarcoma. A673 cells were transfected with Slit2 siRNAs and/or NELL2 siRNAs as indicated. Two days after transfection, the levels of active cdc42 were examined.