Treatment with DZNep depletes expression of polycomb group proteins EZH2, SUZ12, and EED in cultured and primary AML cells. (A) 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 EZH2, SUZ12, EED, and DNMT1. The expression levels of β-actin in the lysates served as the loading control. (B) Primary AML cells were treated with the indicated concentrations of DZNep for 24 hours. At the end of treatment, cell lysates were prepared and immunoblot analysis was performed for EZH2, SUZ12, EED, and DNMT1. The expression levels of β-actin in the lysates served as the loading control. (C) OCI-AML3 cells were treated with BZ and DZNep as indicated for 8 hours. After treatment, cell lysates were prepared and immunoblot analysis was performed for EZH2 and SUZ12. The expression levels of β-actin in the lysates served as the loading control. (D) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 16 hours. Then, total RNA was isolated and quantitative real-time PCR was performed with TaqMan probes for EZH2, SUZ12, and EED. The relative quantity (RQ) of each mRNA was normalized against glyceraldehyde-3-phosphate dehydrogenase expression. (E) HL-60 and OCI-AML3 cells were treated with 2 μM DZNep for 8 and 24 hours. Total RNA was isolated and reverse transcribed with a stem loop primer for hsa-miR-101. After reverse transcription, qPCR for hsa-miR-101 was performed, and expression of hsa-miR-101 was normalized against 18S RNA expression. (F) HL-60 cells were treated with the indicated concentrations of DZNep for 24 hours. Chromatin immunoprecipitation was performed with anti-EZH2 antibody. Immunoprecipitated DNA was used for qPCR of the WNT1, CHD1, and HOXA9 promoters. Fold enrichment data are relative to the control and are expressed as a ratio of the cycle threshold for the chromatin immunoprecipitation DNA versus the cycle threshold for the input samples.