Figure 6.

ISL1 and MYCN act in parallel to regulate common yet distinct oncogenic pathways in neuroblastoma. (A, B) Relative mRNA expression of ISL1, MYCN, GATA3 and selected ISL1 or MYCN downstream genes in ISL1 KD and MYCN KD SK-N-BE(2) neuroblastoma cells. Error bars represent ±SD; n=3; *p<0.05, **p<0.01, 2-tailed t-test. (C-G) EdU staining of control, ISL1 KD, MYCN KD and MYCN/ISL1 double-KD SK-N-BE(2) cells, and quantification of EdU-positive cells. Error bars represent ±SD; ** p<0.01, n=5, 2-tailed t-test. (H-P) neurite outgrowth of ISL1 KD, MYCN KD, ISL1/MYCN-double KD and control SK-N-BE(2) cells with or without RA. All images were taken at 72 hours after RA or DMSO treatment. The images from three biological replicates were pooled, and the length of longest neurite of individual cells was measured (P). In RA untreated groups, the number of measured cells (n) in Ctrl, ISL1 KD, MYCN KD and ISL1/MYCN double-KD group is 100, 98, 95 and 95, respectively. In RA treated groups, the number of measured cells (n) in Ctrl, ISL1 KD, MYCN KD and ISL1/MYCN double-KD group is 105, 121, 96 and 95, respectively. (*p<0.05; **p<0.01; 2-tailed t-test). (Q) The regulatory network controlling neuroblastoma pathogenesis. ISL1, together with GATA3, acts upstream of multiple oncogenic pathway essential for neuroblastoma proliferation and differentiation. In MYCN-amplified SK-N-BE(2) neuroblastoma cells, MYCN acts in parallel with ISL1 to control the cell cycle and RA-mediated differentiation. In addition, MYCN can suppress differentiation (neurite outgrowth) independent of RA pathways.