EZH2 overexpression in NKTL promotes cell growth independent of histone methyltransferase activity. (A) Primary NK cells transduced by EZH2 exhibit a growth advantage. Primary NK cells expressing ectopic EZH2 were monitored by a coexpressed GFP marker. Using our established viral infection protocol, we routinely obtained a transduction efficiency of 4.3% for normal NK cells. If EZH2 infection does not alter the cell growth, the EZH2-infected cell will not gain a growth advantage; thus, the percentage of GFP+ cells should remain at 4.3%. However, the percentage of GFP+ cells increased from approximately 4.3% at day 2 to ∼34.4% at day 5, indicating an acquired growth advantage in these EZH2-infected cells. Antibiotic selection of positively infected cells was not done. (B) Scatterplot representation of the correlation between the percentage of Ki-67–positive cells and the percentage of EZH2-positive cells. Spearman correlation coefficient r for EZH2 v Ki67 = 0.73; P < .0001. (C) MTS proliferation assay showing that ectopic expression of EZH2 promotes cell growth of NKTL cell lines without requiring SET domain activity. Cells were cotransfected with pMAX-GFP and the control empty vector pcDNA4.1 or EZH2 expression plasmids. Cells transfected were subjected to proliferation assays for up to 96 hours. The cell growth (expressed as a percentage of the empty vector control) was determined by MTS assay as described in Materials and Methods. The mean values of triplicate samples are shown, and error bars indicate standard deviations. (D) Western blot analysis of EZH2, H3K27m3, and H3K27m2 in indicated samples. Expression of EZH2 WT and the SET-domain mutant was detected by the MYC-tag antibody. H3 was used as a loading control.