DZNep treatment depletes trimethylation of K27 on histone H3 and induces the expression of cell-cycle regulatory genes p16, p21, and p27, as well as the cell death regulator FBXO32 in AML cells. (A) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 24 hours. After this, nuclear extracts were prepared and immunoblot analysis was performed for 3MeK27 histone H3, 3MeK9 histone H3, 3MeK79 histone H3, and 3MeK4 histone H3. The expression levels of histone H3 in the extracts served as the loading control. (B) OCI-AML3 cells were treated with the indicated concentrations of DZNep for 24 hours. After treatment, total RNA was isolated and RT-PCR was performed for FBXO32, p16, p21, and p27. A β-actin–specific reaction and expression levels served to ensure equal loading. (C) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 24 hours. After this, total cell lysates were prepared and immunoblot analysis was performed for FBXO32, cyclin E, p16, p21, and p27. The expression levels of β-actin in the lysates served as the loading control. (D) OCI-AML3 cells were transfected with scrambled control or EZH2 siRNA for 48 hours. Then, total RNA was isolated and RT-PCR was performed for EZH2, SUZ12, EED, FBXO32, p16, p21, and p27. A β-actin–specific reaction and expression levels served to ensure equal loading. Alternatively, total cell lysates were prepared and immunoblot analysis was performed for EZH2, SUZ12, EED, FBXO32, p16, p21, p27, and 3MeK27 histone H3. The expression level of β-actin in the lysates served as the loading control.