EZH2 depletion inhibits cell growth of NKTL tumor cells. (A) Effects of EZH2 knockdown on cell growth of NKYS cells. EZH2 shRNAs or control shRNA plasmids were cotransfected with a GFP-expressing plasmid pMAX-GFP in cells by electroporation. The percentage of GFP+ cells was determined by a Tali image-based cytometer at 18 hours after transfection. Cells transfected with EZH2 shRNA, but not those transfected with an empty vector or unrelated shRNA, exhibited a severe competitive growth disadvantage and cell death, as indicated by a significant depletion of GFP+ cells with time. (B) qRT-PCR analysis of CCND1 transcription in NKYS cells with EZH2 depleted by RNAi. (C) Rescue of EZH2 shRNA induced cell viability loss by forced expression of EZH2 SETΔ. Endogenous EZH2 was knocked down by an EZH2 shRNA-2, or was restored to physiological levels by ectopically expressing EZH2 shRNA2-resistant EZH2 SETΔ in NKYS and KHYG1 cells. Cells were cotransfected with EZH2 shRNA, together with either EZH2 WT or EZH2 SETΔ. Control transfections included a pLKO shRNA and pcDNA4.1, respectively. pMAX-GFP was cotransfected with other plasmids in each transfection to mark successfully transfected cells. The percentage of GFP+ live cells was determined by a Tali image-based cytometer at 18 hours after transfection. (D) Schematic description of EZH2 protein as well as the relative positions of regions targeted by EZH2 shRNAs. shRNAs expressed from a pLKO.1 vector targeting 3 regions of EZH2 are shown as black bars in relationship to the protein-coding regions.