Figure 6. NELL2, CD133, and EWS-FLI1 positively regulate each other and increase the BAF subunits and cell proliferation in Ewing sarcoma.

(A) Slow growth of the CD133^low population can be rescued by recombinant NELL2. The CD133^low population was treated with the indicated concentration of recombinant NELL2, and cell proliferation was assessed by IncuCyte in comparison with the CD133^high population.

(B) Recombinant NELL2 increases CD133 and BAF subunits in the CD133^low population. The CD133^low population was treated with the indicated concentration of recombinant NELL2 for 4 or 24 h, and the protein levels of CD133, BRG1, BAF250A, BAF155, and BAF47 were assessed by immunoblotting.

(C) NELL2 concentration in the culture supernatant of Ewing sarcoma cell lines. NELL2 concentration in the culture supernatant of 5 Ewing sarcoma cell lines, DMEM medium, and RPMI1640 medium was determined by ELISA (3 independent experiments).

(D) Increasing CD133 in the CD133^low population results in increased NELL2, EWS-FLI1, BRG1, BAF250A, BAF155, and BAF47. The CD133^low population was infected with CD133-expressing lentivirus, and the expression of indicated proteins was assessed by immunoblotting in comparison with uninfected CD133^low population and CD133^high population.

(E) Increasing CD133 in the CD133^low population results in increased cell proliferation. The proliferation of cells in (D) was assessed by IncuCyte.

(F) CD133 silencing results in reduced NELL2, EWS-FLI1, BRG1, and BAF155. A-673, EW8, TC71, and CHLA-9 cells were transfected with CD133 siRNAs (+) or control siRNAs (−), and the expression of indicated proteins was assessed by immunoblotting.

(G) CD133 silencing results in reduced cell proliferation. A-673 cells were transfected with CD133 siRNAs or control siRNAs and cell proliferation was assessed by IncuCyte.

(H) NELL2 silencing results in reduced CD133. A-673 and EW8 cells were transfected with NELL2 siRNAs or control siRNAs, and the levels of NELL2 and CD133 were assessed by immunoblotting.

(I) EWS-FLI1 silencing results in reduced NELL2 and CD133. A-673 and EW8 cells were infected with lentiviruses expressing FLI1 C terminus shRNA (+) or control shRNA (−) and were selected with puromycin. The expression of EWS-FLI1, NELL2, and CD133 was assessed by immunoblotting.

(J) EWS-FLI1 binds to the P2 and P6 promoters of the CD133 gene. Chromatin immunoprecipitation was performed as in Figure 1F (n = 3).

(K) EWS-FLI1 silencing results in reduced CD133 transcript levels in A-673 cells; *p < 0.05 (n = 3).

(L) EWS-FLI1 expression results in increased CD133 transcript levels in human mesenchymal stem cells; *p < 0.05 (n = 3).

(M) NELL2, CD133, and EWS-FLI1 positively regulate one another in Ewing sarcoma.