Fig. 3. Ezh2 is proteasomally degraded via Ube2l6.

A EZH2 expression according to melanoma tumor stage in the Melanoma Research Victoria patient cohort. Stage I (n = 12 patients), Stage II (n = 10 patients), Stage III (n = 9 patients) and Stage IV (n = 8 patients). Data are presented as mean ± SD and analyzed by one-way ANOVA plus Tukey’s multiple comparison test. *p < 0.05, **p < 0.01. B Ezh2 (red) IF in HPCs and LPCs sorted from B16-F10 cells and treated with 10 µM MG132 or DMSO control for 16 h. DAPI (blue): nuclei. Scale bar: 10 µm. C Ezh2 protein levels determined by western blot in B16-F10 cells treated with 10 µM MG132 or DMSO control for 4 h or 8 h. D E2 ligases downregulated in RNAseq data from LPCs vs HPCs. E Ube2l6 IF in LPCs and HPCs from B16-F10 cells. Scale bar: 10 µm. F Ezh2 and H3K27me3 levels were determined by western blot in B16-F10 cells transfected with Flag-tagged Ube2l6-WT, Flag-tagged Ube2l6-C87A (enzyme-dead) or empty vector. (G) As in (F), with or without 10 µM MG132 treatment for 16 h. H Stability of endogenous Ezh2 protein determined by western blot in B16-F10 cells transfected with Flag-tagged Ube2l6-WT or Flag-tagged Ube2l6-C87A, followed by 50 µg/mL CHX treatment for the indicated times. I Densitometry for western blots shown in (H). Data from three independent experiments are presented as mean ± SD and were analyzed by one-way ANOVA plus Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. J Flag-tagged Ube2l6-WT was overexpressed in B16-F10 cells maintained in 10 µM MG132 for 16 h. Interactions between endogenous Ezh2 and Ube2l6 were determined by immunoprecipitation with anti-Ezh2 antibody followed by western blotting with anti-Flag antibody. K HEK293 cells were co-transfected with Flag-tagged EZH2 and Flag-tagged Ube2l6-WT or Flag-tagged Ube2l6-C87A, together with HA-tagged ubiquitin, or (L) HA-tagged ISG15 in the presence of 10 µM MG132. The ubiquitination (K) and ISGylation (L) of EZH2 were determined by anti-HA IP followed by western blot with anti-EZH2 antibody. M 28:B4:F3 and IGR37 cells were infected with V5-tagged empty vector or V5-tagged UBE2L6 lentiviral particles. Positive clones were selected by incubation in 2 µg/µL puromycin for 2 weeks. EZH2 was detected by western blot in stably transfected cells. Representative cell pellets are shown (bottom row). N Clonogenicity of 28:B4:F3 cells assessed by CV staining. O Tumor engraftment in NSG mice harboring control or V5-UBE2l6-WT vector cells 15 weeks after injection. P Volumes of tumors in NSG mice injected with 28:B4:F3 cells harboring control or V5-UBE2l6-WT vector after 15 weeks. The percentages of mice with macro- and micro-metastasis, and number of metastases per mm² to either (Q, S) lung, or (R, T) liver, respectively. U Mean area of micro-metastases in lungs and liver. Control mice number = 11, UBE2L6-OE mice number = 11. Data analyzed by student t-test. *p < 0.05.