Fig. 6.

The NF-κB transactivation-defective mutant, Tax-G148V, exhibits reduced genomic instability and is impaired for p65^RelA-Stathmin molecular interactions. (A) HT1080 cells were transfected with 0.5 mg of the expression constructs for wildtype HTLV-1 Tax or the Tax-G148V mutant, defective for NF-κB transactivation (Yamaoka et al., 1996), and confocal immunofluorescence-microscopy was performed to determine the relative percentages of multinucleate cells per field. Certain samples were also treated with nocodozole (400 ng/ml for 24 hrs). The samples were fixed, permeabilized, and then stained using an Anti-Tubulin primary antibody and rhodamine red-conjugated secondary antibody and DAPI. The data represent the mean ± standard deviation (error bars) from three independent experiments. (B-D) HT1080 cells were cotransfected with 0.5 mg of pcDNA3.1-GFP and the expression constructs for either wildtype HTLV-1 Tax or the Tax-G148V mutant, and the relative percentages of GFP-positive cells with cytoplasmic tubulin aggregates (arrows in C and D) were quantified in-triplicate using confocal immunofluorescence-microscopy. Representative micrographs are shown in C and D. Scale bar, 10 mm. The data in B represent the mean ± standard deviation (error bars) from three independent experiments. (E) The relative levels of acetylated tubulin in nocodozole-treated, untransfected HT1080 cells or transfected cells expressing wildtype HTLV-1 Tax or the Tax-G148V mutant were determined by SDS-PAGE and immunoblotting. Total Alpha-Tubulin protein levels are shown for comparison. (F) 293 HEK cells were transfected with the expression constructs for wildtype HTLV-1 Tax or the Tax-G148V mutant and the Stathmin protein complexed with p65^RelA was co-immunoprecipitated from precleared extracts using Protein G-agarose and a monoclonal Anti-p65^RelA antibody. The relative input levels of Stathmin, p65^RelA, Tax, and Actin are shown in the upper panels. The precipitated Stathmin protein was detected by immunoblotting (lower panel). (G) The relative levels of immunoprecipitated Stathmin in F were quantified by densitometry analysis of the immunoblot bands. All the data in E, F and G are representative of at least three independent experiments; and the data in G represent the mean ± standard deviation (error bars).