Figure 1.
External stimuli and ibrutinib treatment modulate EZH2 expression. (A) EZH2 mRNA analysis using real-time quantitative polymerase chain reaction of 10 CLL cases after BcR stimulation for 12 hours. In 5 of 10 cases, EZH2 was upregulated (FC = 1.7; P < .05), whereas the remaining were downregulated (FC = 1.4; P < .01). In the graph, 2 connected points represent EZH2 relative expression in 2 different conditions for each patient. (B) Each bar in the graph shows the mean values of the FC of EZH2 protein expression (as analyzed by using western blotting) in cells stimulated with CD40L and/or CpG for 12 hours, normalized to unstimulated control cells. Asterisks indicate significant differences compared with the unstimulated control. (C) Immunoblotting analysis of EZH2 protein expression and β-actin of 2 representative cases. (D) Each bar in the graph shows the mean values with standard deviation of EZH2 expression in CLL cells alone or CLL cells cocultured with HS-5 cells for 3 days (n = 4, FC = 1.5; P < .05). CLL cell viability analysis (E) and H3K27me3 levels (F) at day 3 after TLR9 stimulation, using flow cytometry. In the graph, connected points represent the percentage of viable cells or H3K27me3 levels in 2 conditions: unstimulated control (CTL) and the cells stimulated with CpG (n = 6; FC = 2.1, P < .01; FC = 1.2, P < .05). (G) Each bar in the graph shows the mean values of FC of Bcl-xl, Mcl-1, cleaved PARP, and cleaved caspase-3 of stimulated CLL cells with CpG for 3 days, compared with unstimulated control cells. Asterisks indicate significant differences compared with the unstimulated control (n = 4, FC = 4.8, P < .01; FC = 3.7, P < .05; FC = –1.7 and FC = –2, respectively). (H) CD19+ cells from patients before treatment initiation with ibrutinib and at 1, 6, and >6 months under treatment were lysed for western blotting analysis of EZH2 protein expression. Each bar in the graph represents the median values with range of EZH2/β-actin protein expression normalized to the pretreatment sample (n = 9, FC = 4.3, P < .001). (I) Immunoblotting analysis of EZH2/β-actin levels for 1 representative case. *P < .05, **P < .01, ***P < .001.  IgM, immunoglobulin M. 

External stimuli and ibrutinib treatment modulate EZH2 expression. (A) EZH2 mRNA analysis using real-time quantitative polymerase chain reaction of 10 CLL cases after BcR stimulation for 12 hours. In 5 of 10 cases, EZH2 was upregulated (FC = 1.7; P < .05), whereas the remaining were downregulated (FC = 1.4; P < .01). In the graph, 2 connected points represent EZH2 relative expression in 2 different conditions for each patient. (B) Each bar in the graph shows the mean values of the FC of EZH2 protein expression (as analyzed by using western blotting) in cells stimulated with CD40L and/or CpG for 12 hours, normalized to unstimulated control cells. Asterisks indicate significant differences compared with the unstimulated control. (C) Immunoblotting analysis of EZH2 protein expression and β-actin of 2 representative cases. (D) Each bar in the graph shows the mean values with standard deviation of EZH2 expression in CLL cells alone or CLL cells cocultured with HS-5 cells for 3 days (n = 4, FC = 1.5; P < .05). CLL cell viability analysis (E) and H3K27me3 levels (F) at day 3 after TLR9 stimulation, using flow cytometry. In the graph, connected points represent the percentage of viable cells or H3K27me3 levels in 2 conditions: unstimulated control (CTL) and the cells stimulated with CpG (n = 6; FC = 2.1, P < .01; FC = 1.2, P < .05). (G) Each bar in the graph shows the mean values of FC of Bcl-xl, Mcl-1, cleaved PARP, and cleaved caspase-3 of stimulated CLL cells with CpG for 3 days, compared with unstimulated control cells. Asterisks indicate significant differences compared with the unstimulated control (n = 4, FC = 4.8, P < .01; FC = 3.7, P < .05; FC = –1.7 and FC = –2, respectively). (H) CD19+ cells from patients before treatment initiation with ibrutinib and at 1, 6, and >6 months under treatment were lysed for western blotting analysis of EZH2 protein expression. Each bar in the graph represents the median values with range of EZH2/β-actin protein expression normalized to the pretreatment sample (n = 9, FC = 4.3, P < .001). (I) Immunoblotting analysis of EZH2/β-actin levels for 1 representative case. *P < .05, **P < .01, ***P < .001.  IgM, immunoglobulin M. 

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