Fig. 7: USP3 is responsible for ALYREF-driven MYCN ubiquitination.

a Immunoblot analysis of MYCN and USP3 expression in Kelly and SK-N-BE(2)C cells following siRNA-mediated USP3 knockdown for 48 h. b Cell viability (n = 3 per group) (c) and proliferation (n = 3 per group) analysis of Kelly and SK-N-BE(2)C cells transfected with USP3 siRNA-1, USP3 siRNA-2, or Control siRNA for 96 h. Two-sided unpaired Student’s t-tests were performed to derive p-values. Differences in cell growth were compared to Control siRNA expressing cells. d Kelly and SK-N-BE(2)C cells transfected with USP3 siRNA-1, USP3 siRNA-2, or Control siRNA were grown for 12 days (Kelly) or 10 days (SK-N-BE(2)C), followed by colony formation assay. e Immunoblot analysis of USP3 expression in stable Kelly and SK-N-BE(2)C cells overexpressing USP3 (USP3) or Vector control (Vector). NTC, non-transduced control cells. f Stable Kelly and SK-N-BE(2)C cells overexpressing USP3 (USP3) or Vector control (Vector) were co-transfected with ALYREF siRNA-1 or ALYREF siRNA-2 or Control siRNA, followed by cell viability measurements (n = 3 per group) at 72 h. Two-sided unpaired Student’s t-tests were performed to derive p-values. Differences in cell viability were compared to Vector Control siRNA expressing cells. g Stable Kelly and SK-N-BE(2)C cells overexpressing USP3 (USP3) or Vector control (Vector) were co-transfected with ALYREF siRNA-1, or ALYREF siRNA-2 or control siRNA, followed by treatment with MG132 for 4 h. Cells were then subjected to endogenous MYCN immunoprecipitation and immunoblot analyses for K-63 and K-48-linked ubiquitination. Respective p-values (p) are displayed. Comparisons were not significant unless otherwise noted. Data are representative of three independent experiments with similar results in a, d, and e. Data are representative of two independent experiments with similar results in g. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in b, c, and f.