Figure 3

Tax expression induces DDSBs by stimulating intracellular NO. HR activity (A) is reduced in 289 cells when compared to BRCA1 cells. Tax triggers H2AX phosphorylation in BRCA1-deficient cell. The 289 and BRCA1 cells were infected with GFP or Tax-IRES GFP-expressing virus during 48 hours. 293T cells were transfected with 1.5 μg DR-GFP and 0.5 μg pSCE I expression vector or empty vector. The percentage of GFP+ cells was estimated by FACS. (B) Tax triggers H2AX phosphorylation in BRCA1-deficient cell. The 289 and BRCA1 cells were infected with GFP or Tax-IRES GFP-expressing virus during 48 hours. The cells were analyzed (B) by Flow cytometry and (C) by western blot. (D-F) Tax and M47 Tax mutant stimulate NO production and trigger H2AX phosphorylation. 293T cells were transfected with 2 μg of empty vector (Ø), Tax, or Tax mutants (M47 and G148V). At 72 h, cell culture supernatant was harvested and tested for NO production (D), and cells were further analyzed by FACS for ɣ-H2AX quantification (E) and by western blot for Tax expression (F). (G-I) The NOS inhibitor L-NMMA blocks NO production and DDSBs induced by Tax. 293T cells were transfected with the empty vector (Ø), Tax, or Tax mutants (M47 and G148V) during 24 h and treated with DMSO or 30 μM L-NMMA for 48 h. Cell culture supernatant was harvested and tested for NO production (G). Cells were analyzed by FACS for ɣ-H2AX quantification (H) and western blot to confirm Tax expression (I).