DHMEQ inhibits constitutive NF-κB activity and induces apoptosis of primary ATL cells. (A) EMSA and supershift analyses. (Left upper 3 panels) DHMEQ inhibition of constitutively activated NF-κB in 3 samples of primary ATL cells. Cells were treated with or without 10 μg/mL DHMEQ for 16 hours. Positions of shifted bands and free probes are indicated on the left. (Left lower 3 panels) Results of EMSA with AP-1 probe. (Right 3 panels) Supershift analysis of NF-κB components in 3 samples of primary ATL cells. Nuclear extracts of ATL samples were subjected to supershift analysis with antibodies specific for p50, p65, c-Rel, p52, and RelB. (B) EMSA of DHMEQ effects on T cell-enriched normal PBMCs and supershift analysis of NF-κB components. (Left panel) Nuclear extracts were prepared after 16 hours of treatment with or without 10 μg/mL DHMEQ. Results of control EMSA with an OCT1 probe are shown at the bottom. (Right panel) Supershift analysis of NF-κB components using antibodies indicated above the gel. The position of shifted bands is indicated on the left. Antibodies used are indicated on the top. (C) Effects of DHMEQ on the viability of primary ATL cells. Cells were treated with 10 μg/mL DHMEQ for 48 hours. Cell viability was measured by MTT assay, and the relative levels compared with those of DMSO-treated cells are presented. Data represent the mean ± SD of 3 independent experiments. (D) Detection of apoptosis by annexin V. Three ATL samples and normal PBMCs were treated with 10 μg/mL DHMEQ, and binding of FITC-conjugated annexin V was analyzed by flow cytometry after 24 or 48 hours. Data represent the mean ± SD of 3 independent experiments. (E) Changes in the nuclear morphology by DHMEQ treatment. Primary ATL cells and control PBMCs were treated with 10 μg/mL DHMEQ for 24 hours and stained with Hoechst 33342. Original magnification ×400. Objective lens, 60×/4.4.