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Figure 5 | Cell & Bioscience

Figure 5

From: Modulation of the stability and activities of HIV-1 Tat by its ubiquitination and carboxyl-terminal region

Figure 5

Tat-induced apoptosis is modulated by its carboxyl-terminal region. (A) HeLa cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone. 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). 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. (D) Quantification of the results in (C). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector 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 collected 96 hours post-transfection. Cell lysates were subjected to immunoblot analysis with antibodies specific for cleaved caspase-3, α-tubulin, or GFP, respectively. (F) 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. (G) Quantification of the results in (F). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector 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. Cropped blots are used in this figure, and the gels were run under the same experimental conditions.

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