Figure 4

Tat-induced apoptosis is regulated by its ubiquitination. (A) HeLa cells were cotransfected with GFP-Tat101 and His-Myc-tagged WT ubiquitin or the lysine mutants. 72 hours post-transfection, cells were treated with (+) or without (-) MG132 and incubated for additional 24 hours. Apoptotic cells were examined via the fluorescence microscope by analyzing cell morphology of GFP-positive cells. (B) Quantification of the results in (A). The number of apoptotic cells was counted and the percentage of apoptotic cells was calculated. Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated ubiquitin-K0 mutant transfection group. Experiments were done in duplicate, six fields per group were counted, and the results were expressed as the mean ± SD. (C) HeLa cells were transfected and treated as in (A), and the apoptotic cells were examined by Annexin V-APC staining coupled with flow cytometry. Mock transfected HeLa cells without Annexin V-APC staining were used as negative control (NC). (D) Quantification of the results in (C). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated ubiquitin-K0 mutant transfection group. Mean and standard deviations were derived from two independent experiments done in duplicate. (E) HeLa cells were transfected and treated as in (A), and stained with anti-cleaved caspase-3 antibodies and the DNA dye DAPI. Apoptotic cells were indicated by hollow arrows. (F) Quantification of the results in (E). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated ubiquitin-K0 mutant transfection group. Experiments were done in duplicate, 80 GFP-positive cells per group were counted, and the results were expressed as the mean ± SD. Two-tailed Student’s t-test for all graphs. *P < 0.05, **P <0.01, ***P < 0.001; ns, not significant.