Fig. 3

EZH2 inactivation by GSK126 induces apoptosis and abrogates outgrowth of UM tumor. a-b GSK126 induced apoptosis in UM cells. UM cells were treated with the indicating concentrations of GSK126 for 24 h, followed by staining with Annexin V-FITC and PI for flow cytometer analysis. A set of representative dot plots (left) and bar charts of annexin-V⁺ cell percentage (middle and right) from 3 independent experiments were shown. The y-axis represents the sum of the top right and bottom right quadrants. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons (a). Western blotting analysis of PARP cleavage, activation of caspase-9, − 8, − 3 and apoptosis-related proteins in GSK126 treated UM cells were shown (b). c-e Survivin mediated GSK126-induced cell apoptosis in UM. Stably expressed with plasmids or shRNAs as indicated, 92.1 and Mel270 cells were incubated with GSK126 for 24 h, followed by cell death detection. Western blotting analysis of cleaved caspase-3 (c) and Trypan blue exclusion assay were applied (d and e). **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons. ##, P < 0.01; ###, P < 0.0001, Student’s t test. f-g GSK126 abrogated outgrowth of xenografted Omm1 tumor and MP41 PDX in NOD/SCID mice. The estimated tumor volumes were plotted against the day of treatment. The last measurements of the 2 groups were compared (f). Weight of tumors dissected from the mice treated with placebo and GSK126 was compared (g). ***, P < 0.0001, Student’s t test