NF-κB–mediated activation of the hTERT promoter. (A) Schematic representation of the hTERT luciferase reporter construct P-330. (B) P-330 (1 μg) was transfected into 293 T cells in the absence (HRCMV, empty vector) or in the presence of increasing amounts of cytomegalovirus (CMV) Tax or CMV Tax mutants M22, G148V, and M47 (0.1, 0.3, and 0.6 μg) and TK-Renilla (60 ng) using the Effectene Reagent (Qiagen). Dual luciferase assay was performed 36 hours later. Normalized results using Renilla values are representative of 2 independent experiments. Experimental variations are indicated by error bars. (C) Western blot analysis for relative expression of Tax and Tax mutants in transfected 293 T cells. (D) P-330 (1 μg) was transfected into 293T cells in the absence or presence of Tax (0.25 μg) with or without I-κBα mutant (50 ng), and luciferase assays were performed as described in “Materials and methods.” (E) Tax expression, detected by Western blot, was not affected by I-κBα mutant expression. (F) HTLV-I–LTR luciferase (1 μg) was transfected into 293T cells in the absence or presence of Tax (0.25 μg) with or without I-κBα mutant (50 ng), and luciferase assays were performed as described in “Materials and methods.” Experimental variations are indicated by error bars. (G) Southern blot analysis of telomere length in Tax-transduced PBMCs and G148V-transduced PBMCs after 10 weeks of culture in RPMI 1640, 20% FBS, and IL-2. Ethidium bromide gel showing high–molecular weight DNA used in telomere length analysis. TRF indicates telomere restriction length fragments.