Figure 1
Tax induces G 1 cell cycle arrest and apoptosis. HeLa cells were transiently transfected with a pCAGGS-Tax Flag-tagged vector or the control pCAGGS vector (A, B and E) together with either the reporter plasmid pGV-HL21 (HTLV-1 enhancer) and the reference plasmid pRL-SV40 (C), or the GFP expression vector pEGFP-NI (D and G) or the pSV-β-galactosidase vector (F). (A) At 24 h post-transfection, cells were lysed and subjected to immunoblot analysis with an anti-Flag MAb and an anti-actin MAb (as a control). (B) At 24 h post-transfection, cells were fixed, permeabilized, and immunostained with an anti-Flag MAb followed by an Alexa 488-conjugated anti-mouse IgG antibody. Cells were analyzed by confocal laser scanning microscopy (Olympus FV1000). (C) At 48 h after transfection, cells were recovered and the activities of firefly and Renilla luciferases were measured in lysates. For each sample, the firefly luciferase activity (pGV-HL21) was normalized by reference to Renilla luciferase activity (pRL-SV40). (D) At 48 h post-transfection, cells were fixed and stained with propidium iodide for the analysis of DNA content. GFP-positive cells were analyzed by flow cytometry using Cell Quest for acquisition and ModFit LT. The peaks of the cells at G1 and G2/M phase are indicated. (E) At 48 h post-transfection, cells were collected, lysed, and analyzed for phosphorylation of Rb by immunoblotting with an anti-Rb MAb using an anti-actin MAb as a control. ppRb, hyperphosphorylated forms of Rb; pRb, hypo- and unphosphorylated forms of Rb. (F) At 48 h post-transfection, cells were collected, lysed, β-galactosidase activity was measured. Caspase-3 activity was measured in the cell lysates with an equal amount of β-galactosidase activity. Each of the columns and its associated error bar represent the mean ± standard deviation (SD) of results from four different experiments. The asterisk (*) represents a p-value of < 0.01. (G) At 48 h post-transfection, cells were stained with PE-Annexin V and 7-AAD to identify apoptotic cells. GFP was used as a reporter to discriminate between transfected and untransfected cells. The percentage of Annexin V-positive and 7-AAD-negative cells relative to GFP-positive cells indicates the level of apoptosis.