Figure 3. NELL2 signaling downregulates cdc42 and upregulates the BAF complexes.

(A) Silencing of srGAPs inhibits Ewing sarcoma proliferation. srGAP1 and srGAP2 were silenced by siRNAs, and cell proliferation was assessed by IncuCyte (left). The silencing of srGAP1 and srGAP2 was verified by immunoblotting (right).

(B) NELL2 silencing or Robo3 silencing increases filopodia in Ewing sarcoma cells. The effect of NELL2 silencing or Robo3 silencing on actin cytoskeleton was assessed by phalloidin staining (red). Cell nuclei were stained by DAPI (blue). The quantification of filopodia using the filopodia image analysis software (Saha et al., 2016) is shown at the bottom. For each sample, 10 randomly chosen fields containing a total of 100–250 cells were analyzed. *p < 0.05 compared with control siRNA transfected cells. Scale bars: 10 μm.

(C) NELL2 silencing activates cdc42 and Rac in Ewing sarcoma. A-673 and EW8 cells were transfected with NELL2 siRNAs or control siRNAs. Two days after transfection, the levels of GTP-bound, active cdc42, Rac, and RhoA were examined by GST-PAK1 (for cdc42 and Rac1) or GST-Rhotekin-RBD (for Rho A) pull-down of whole-cell lysate followed by anti-cdc42, Rac, and Rho A immunoblotting.

(D) The regulation of cdc42 and Rac activities by NELL2 requires srGAPs.

(E) A selective cdc42 inhibitor, ML141, abrogates the proliferation inhibition by NELL2 silencing or Robo3 silencing in Ewing sarcoma. A-673 and EW8 cells were transfected with NELL2 siRNAs, Robo3 siRNAs, or control siRNAs and were treated with or without 5 μM ML141. Cell proliferation was assessed by IncuCyte.

(F) cdc42 silencing abrogates the proliferation inhibition by NELL2 silencing. The silencing of NELL2 and cdc42 was verified by immunoblotting (right).

(G) NELL2 signaling inhibits cdc42, the normal function of which is to promote filopodia formation and inhibit cell proliferation in Ewing sarcoma.

(H) NELL2 silencing reduces EWS-FLI1 target gene expression in Ewing sarcoma. A-673 and EW8 cells were transfected with NELL2 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 NELL2-silenced cells (red) (n = 3).

(I) NELL2 silencing reduces the protein levels of some of the BAF subunits in Ewing sarcoma. Ewing sarcoma cells were transfected with NELL2 siRNAs or control siRNAs, and the levels of indicated BAF subunits were examined by immunoblotting.

(J) Recombinant NELL2 restores the protein levels of BAF subunits in NELL2-silenced cells. A-673 and EW8 cells were transfected with NELL2 siRNAs or control siRNAs and were treated with recombinant NELL2 (250 ng/mL) for the indicated time (hours). The levels of BAF subunits were assessed by immunoblotting.

(K) NELL2 signaling upregulates the BAF complexes and enhances the transcriptional output of EWS-FLI1.