DHMEQ inhibits constitutive NF-κB activity in HTLV-1-transformed and ATL-derived cell lines. (A) EMSA of NF-κB. Inhibition of constitutive NF-κB binding activity by DHMEQ in HTLV-1-transformed and ATL-derived cell lines (left 3 panels). A myeloid leukemia cell line K562 without HTLV-1 infection was used as a control. Cells were cultured with or without 10 μg/mL DHMEQ for 16 hours. Nuclear extracts from Jurkat cells treated with TNF-α served as a control. Upper panels show inhibition of NF-κB binding activity by DHMEQ. Lower panels show results of EMSA with control probes, AP-1 and OCT1. The OCT1 probe was used for HTLV-1-uninfected cells that do not show constitutive activation of AP-1. (B) Inhibition of NF-κB transcription activities in ATL-derived cell lines by DHMEQ. Relative levels of luciferase activities are shown in percentages compared with the levels of untreated cells. M indicates MT-1 cells; T, TL-Om1 cells; NF-κB, NF-κB-driven luciferase construct; AP-1, AP-1-driven luciferase construct. Renilla luciferase vector (pRL-TK) was used to standardize the transfection efficiency. (C) Supershift analysis of the NF-κB components in HTLV-1-transformed and ATL-derived cell lines. Antibodies used are indicated on top. The position of a shifted band corresponding to NF-κB is indicated on the left. (D) Inhibition of nuclear translocation of NF-κB by DHMEQ. Representative results of confocal immunofluorescence analysis using antibodies against NF-κB p65 or NF-κB p50. Original magnification ×600. Images captured with a60×/1.4 objective lens.