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. Author manuscript; available in PMC: 2018 May 3.
Published in final edited form as: Cell Rep. 2018 Apr 17;23(3):823–837. doi: 10.1016/j.celrep.2018.03.078

Figure 6. A ZRANB1 Inhibitor Destabilizes EZH2 and Inhibits Cell Viability through ZRANB1.

Figure 6

(A) Purified His-ZRANB1 was pretreated with NSC60650 or NSC112200 for 10 min and then incubated with K33-linked di-ubiquitin in the presence of the compound at 37°C for 1.5 hr. Samples were then analyzed by immunoblotting with a ubiquitin-specific antibody.

(B) Immunoblotting of EZH2 and β-actin in LM2 cells treated with NSC112200 at the indicated doses for 24 hr.

(C) LM2 cells were treated with NSC112200 at the indicated doses. 24 hr after treatment, viable cells were quantitated by an MTT assay. n = 4 biological replicates.

(D) Immunoblotting of EZH2 and β-actin in BT549 cells treated with NSC112200 at the indicated doses for 24 hr.

(E) BT549 cells were treated with NSC112200 at the indicated doses. 24 hr after treatment, viable cells were quantitated by an MTT assay. n = 4 biological replicates.

(F and G) Immunoblotting of EZH2, SUZ12, and β-actin in LM2 (F) and BT549 (G) cells treated with NSC60650 or NSC112200 at the indicated doses for 24 hr.

(H) Immunoblotting of EZH2 and β-actin in LM2 cells pretreated with 2 μM MG132 for 1 hr and then treated with 10 μM NSC112200 in the presence of MG132 overnight.

(I) HEK293T cells were co-transfected with MYC-EZH2 and HA-ubiquitin (Ub), pretreated with 2 μM MG132 for 1 hr, and then treated with 10 μM NSC112200 in the presence of MG132 overnight, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and EZH2. N.S., non-specific signal.

(J) Left panel: immunoblotting of EZH2 and β-actin in control and ZRANB1-knockout HEK293A cells treated with 10 μM NSC112200 overnight. Right panel: quantification of EZH2 protein levels (normalized to β-actin) is shown.

(K) Control, ZRANB1-knockout, and ZRANB1-restored HEK293A cells were treated with 10 μM NSC112200 for 24 hr, and viable cells were quantitated by an MTT assay. Data are normalized to vehicle-treated cells for each group. n = 4 biological replicates.

Error bars in (C), (E), and (K) are SEM; p values were calculated from a two-tailed t test. See also Figure S6.