Fig. 4: EZH2 regulates MYCN stabilization independent of its methyltransferase activity.

a Analysis of MYCN polyubiquitination in the presence of WT EZH2 or the methyltransferase-inactive mutant (H689A). 293T cells were transfected with plasmids expressing epitope-tagged MYCN, ubiquitin, FBW7α and WT or H689A EZH2 for 48 h as shown. Ubiquitin-conjugated MYCN proteins were pulled down with SBP beads and subjected to immunoblot with ubiquitin antibody. b Comparison of MYCN protein levels in 293T cells expressing EZH2 WT or H689A. 293T cells were transfected with plasmids expressing epitope-tagged MYCN, FBW7α, and EZH2 (WT or H689A) for 48 h as shown. MYCN and other indicated proteins were analyzed by immunoblots. c Kelly cells upon shRNA depletion of endogenous EZH2 (targeting EZH2 3′UTR) were transduced with EZH2 WT or H689A. MYCN and other indicated proteins were analyzed by immunoblots. Histone H3 was used as a loading control. d Immunoblot of MYCN in the presence of various EZH2 inhibitors. Kelly cells were respectively treated with 5 μM of DZNep, GSK126, or EPZ6438 for 48 h before subjected to immunoblotting. e Co-immunoprecipitation (co-IP) to detect the protein-protein between MYCN and FBW7αΔF in the presence of EZH2 WT and H689A. 293T cells were transfected with plasmids expressing epitope-tagged MYCN, FBW7αΔF (F-box deletion mutant) and increasing amounts of EZH2 WT or H689A mutant for 48 h as shown. HA-MYCN-associated proteins were analyzed by immunoblot. f In vitro pull-down experiments to detect the protein–protein between MYCN and EZH2 (or FBW7α). Recombinant His-tagged MYCN was incubated with GST-tagged FBW7α and/or increasing amounts of EZH2. MYCN-associated proteins were precipitated by nickel beads and analyzed by immunoblotting. The experiments were independently repeated three times with similar results (a–f). Source data are provided as a Source data file.