Fig. 3.

HTLV-1 Tax specifically induces the activation of iNOS expression. a Tax inducible cell lines JPX9 and TetOn-Tax were induced for 48 h with CdCl₂ (40 μM) and doxycycline (500 ng/ml) for 24 and 48 h respectively, to express the Tax protein. Cell extracts were subject to Western blot analysis using antibodies against iNOS, Tax and actin. b Doxycyline-induced TetOn-Tax cells were left untreated or treated with 1400 W (7 nM) for 48 h. Western blots were performed on the cell extracts by using anti-p-H2AX, anti-pChk2, anti-H2AX, anti-Chk2, and anti-iNOS antibodies. The percentage of expression of DNA damage response after iNOS inhibition was quantified by the ratio of phosphoprotein on the total amounts of proteins, and it was indicated underneath the Western blot. * non-specific band. c NO staining with DAF-2DA was performed on two experiments in which TetOn-Tax was induced and cells treated with iNOS inhibitor, 1400 W, to show that the reduction in DNA damage response was associated with a relative decrease in NO production. d, e Real time PCR of iNOS and IκBα mRNA in negative control CD4⁺ and Jurkat cells, in HTLV-1 transformed cell lines MT4 and HuT102, and in three Tax-negative ATL cell lines, MT1, ED4505- and TL-Oml1.