Figure 9. EZH2-regulated gene IGFBP3 functions as a neuroblastoma tumor suppressor.

(A) The protein expression level of IGFBP3, a representative gene in the neuroblastoma EZH2 signature, in human neuroblastoma cell lines with or without MYCN amplification. (B) EZH2, H3K27me3, and H3K4me3 binding signal at the promoter of IGFBP3 in Kelly cells. (C) Immunoblot showing the overexpression of EGFP (negative control), NGFR (positive control), and IGFBP3 in SK-N-BE(2) cell line. (D) Cell viability assay after overexpression of EGFP, NGFR, or IGFBP3 in SK-N-BE(2). Results are representative of 3 independent experiments; mean ± SD of 8 technical replicates is shown. (E) Tumor volume in mouse xenograft model of SK-N-BE(2) with or without IGFBP3 overexpression (n = 10). P calculated with 2-way ANOVA. (F) Kaplan-Meier curves show survival of mice with xenografts of SK-N-BE(2) with or without IGFBP3 overexpression, up to 56 days after injection. P calculated using log-rank (Mantel-Cox) test. (G and H) Cell viability assay (G) and immunoblotting (H) after overexpression of IGFBP3 in CHP-212, LAN-1, ACN, and SH-SY-5Y. Results are representative of 3 independent experiments; data in G represent mean ± SD of 8 technical replicates. (I) Effect of overexpression of IGFBP3 on SK-N-BE(2)’s response to EZH2 inhibitors. Shown is a representative of 2 independent experiments; mean ± SD of 8 technical replicates is shown.