Fig 1: SS18-SSX translocation positive synovial sarcoma cell lines are sensitive to EZH2 inhibition in vitro.(A) Immunoblot analysis of human soft tissue sarcoma (RD and SW982), human synovial sarcoma (Fuji and HS-SY-II), human MRT (G401), and human embryonic kidney (293) cell lines to examine expression of EZH2, SMARCB1, SS18/SS18-SSX, and ß-actin (loading control). Closed and open arrowheads represent SS18–SSX and SS18, respectively. (B) The synovial sarcoma cell lines were treated at increasing doses of tazemetostat for 14 days and proliferation was assessed at the indicated timepoints. Tazemetostat was refreshed on days 4 and 11. Data represent the geometric mean ± 95% confidence interval in triplicate from 3 independent experiments. IC50, 50% inhibitory concentration value. (C) H3K27Me3 levels were assessed in HS-SY-II, Fuji and SW982 treated with tazemetostat for 96 hours. Levels were quantified using ELISA and data is represented as the ratio of H3K27Me3 to total H3 and are shown relative to the DMSO control of each concentration. Data are represented as mean values ± SEM (n = 3). (D, E) Apoptosis analysis (Annexin V-FITC assay) (D) or cell-cycle analysis (by flow cytometry) (E) of Fuji and HS-SY-II treated with the IC50 for tazemetostat (0.15 µmol/L and 0.52 µmol/L, respectively). Data are represented as mean values ± SEM (n = 3). For panel D: * P< 0.05, ** P<0.01, *** P<0.001, **** P<0.0001, vs. untreated, one-way analysis of variance followed by the Dunnett’s multiple comparisons test.