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Figure 3


Fig. 3. Mutational inactivation of the C-terminal NTS of TERT completely disrupts TERT nucleolar localization in both normal and malignant human cells. Wild-type GFP-TERT and the mutant GFP–TERT-3A, which contains the substitution mutations indicated in Fig. 2A, were individually introduced, through recombinant lentiviral infection, into BJ cells (normal human fibroblasts) or into HeLa, H1299 or U2OS cancerous cells for stable expression. (A) A representative example showing that ectopically overexpressed GFP-TERT (green, upper panel) concentrates within nuclear foci identical to the nucleolar structures observed under optic microscope observation. However, overexpressed GFP–TERT-3A (green, lower panel) distributed diffusely in the nucleoplasm, outside nucleoli, in stable BJ cells. 100x magnification. (B) Images showing that overexpressed GFP-TERT exhibited a predominant nuclear diffusion pattern in all indicated transfected cancer cell lines and that GFP–TERT-3A displayed a predominant nucleolar-exclusion pattern in these cell lines. 400x magnification. Arrows indicate nucleolar regions identified under optic microscope observation. The percentages of cells with each representative image in the corresponding cell populations are presented to the right.





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