Figure 4
Figure 4. Regulatory mechanism of EZH2 expression. (A-B) Relationship between the levels of EZH2 and epigenetically suppressed gene set. ATL patient samples were divided into 2 groups according to EZH2 level. Box plots show expression levels of EZH2 (A) and H3K27me3 target genes (B) in normal CD4+ T cells (n = 21) and ATL cells (EZH2 low, n = 25; EZH2 high, n = 25) (*P < .001). (C) EZH2 and H3K27me3 levels in TL-Om1 with shcontrol or shEZH2 analyzed by western blotting. (D) EZH2 expression level in CD4+ T cells that were unstimulated, stimulated with anti-CD3 antibody, or stimulated with anti-CD3/CD28 antibodies for 48 hours. (E) EZH2 expression level in CD4+ T cells that were unstimulated or stimulated with polymethacrylate and/or ionomycin for indicated times. (F) EZH2 mRNA level in CD3+ T cells that were unstimulated or stimulated with anti-CD3/CD28 antibodies or tumor necrosis factor-α in the presence or absence of pretreatment (2 hours before stimulation) with NF-κB or NFAT inhibitor (n = 3, mean ± SD, P < .05). (G) EZH2 level in MT-1 cells that were treated with intracellular signaling inhibitor series (10 μM BMS345541, 20 μM LY294002, 20 μM SP600125, 20 μM SB203580, 20 μM U0126, 1 μg/mL cyclosporin A) for 48 hours. DZNep (5 μM) was used as a positive control.49 (H) mRNA levels of PRC2 core components in MT-1 and TL-Om1 cells in the presence or absence of IKKβ inhibitor BMS345541 for 12 hours (n = 3, mean ± SD, P < .05). (I) Western blots showing PRC2 core component levels in primary ATL samples that were treated with BMS345541 (5 or 10 μM) and DHMEQ (10 μg/mL) for 48 hours. (J) PCR-ChIP assay detected RelA and RelB bindings on EZH2 promoter region in TL-Om1 cells. Promoter regions of GAPDH and BCL-XL genes were used as a negative or a positive control, respectively. (K) EZH2-promoter activity in the presence or absence of IKKβ inhibitor BMS345541 for 12 hours analyzed by dual-luciferase reporter assay (n = 4, mean ± SD, P < .05). The role of putative NF-κB binding sites was evaluated by mutant reporters (indicated by open triangles).

Regulatory mechanism of EZH2 expression. (A-B) Relationship between the levels of EZH2 and epigenetically suppressed gene set. ATL patient samples were divided into 2 groups according to EZH2 level. Box plots show expression levels of EZH2 (A) and H3K27me3 target genes (B) in normal CD4+ T cells (n = 21) and ATL cells (EZH2 low, n = 25; EZH2 high, n = 25) (*P < .001). (C) EZH2 and H3K27me3 levels in TL-Om1 with shcontrol or shEZH2 analyzed by western blotting. (D) EZH2 expression level in CD4+ T cells that were unstimulated, stimulated with anti-CD3 antibody, or stimulated with anti-CD3/CD28 antibodies for 48 hours. (E) EZH2 expression level in CD4+ T cells that were unstimulated or stimulated with polymethacrylate and/or ionomycin for indicated times. (F) EZH2 mRNA level in CD3+ T cells that were unstimulated or stimulated with anti-CD3/CD28 antibodies or tumor necrosis factor-α in the presence or absence of pretreatment (2 hours before stimulation) with NF-κB or NFAT inhibitor (n = 3, mean ± SD, P < .05). (G) EZH2 level in MT-1 cells that were treated with intracellular signaling inhibitor series (10 μM BMS345541, 20 μM LY294002, 20 μM SP600125, 20 μM SB203580, 20 μM U0126, 1 μg/mL cyclosporin A) for 48 hours. DZNep (5 μM) was used as a positive control.49  (H) mRNA levels of PRC2 core components in MT-1 and TL-Om1 cells in the presence or absence of IKKβ inhibitor BMS345541 for 12 hours (n = 3, mean ± SD, P < .05). (I) Western blots showing PRC2 core component levels in primary ATL samples that were treated with BMS345541 (5 or 10 μM) and DHMEQ (10 μg/mL) for 48 hours. (J) PCR-ChIP assay detected RelA and RelB bindings on EZH2 promoter region in TL-Om1 cells. Promoter regions of GAPDH and BCL-XL genes were used as a negative or a positive control, respectively. (K) EZH2-promoter activity in the presence or absence of IKKβ inhibitor BMS345541 for 12 hours analyzed by dual-luciferase reporter assay (n = 4, mean ± SD, P < .05). The role of putative NF-κB binding sites was evaluated by mutant reporters (indicated by open triangles).

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