Fig. 6

Combined targeting of PRC2 and class I HDACs results in the inhibition of proliferation and tumor growth. a Proliferation analysis of EwS cell lines CHLA-10, EW7 and SK-N-MC treated with either A-395, FK228 or a combination of both. Cell impedance was measured every 4 h. Data are shown as mean ± SEM (hexaplicates/group; p-value < 0.0001). b In vivo antitumor efficacy of FK228 and A-395 as a single agent or in combination against tumor-bearing Rag2^−/−γ_C^−/−mice. 2 × 10⁶ CHLA-10 or SK-N-MC EwS cells were injected subcutaneously (s.c.) into mice. Once tumors were palpable mice were randomly allocated into four groups (5 mice/group) and treated with vehicle control (DMSO), A-395 (250 mg/kg, s.c. twice per week), FK228 (2 mg/kg, intraperitoneal (i.p.) once per week), or with A-395 in combination with FK228 for 4 weeks (p < 0.0001). c All tumors were analyzed for cleaved caspase 3 by immunohistochemistry. Representative results of CHLA-10 tumors are shown (10x original magnification). d Top, the level of cleaved caspase 3 in CHLA-10 and SK-N-MC derived tumors. The percentage of cleaved caspase 3 positive cells in five fields per tumor is given. Bottom, the number of mitoses per 10 high power fields per tumor is given. e Interaction analysis by Co-IP of PRC2 protein EED with HDAC1, and HDAC2. Co-IP for CHLA-10, EW7, and SK-N-MC cell lines using anti-EED antibodies. After Co-IP, proteins were analyzed by western blotting for HDAC1, 2, 3 and 8. GAPDH served as a loading control