FIG. 3.
Nuclear export of hTERT occurs through the nuclear pores. (A) A representative immunostaining is shown for 293 cells overexpressing full-length, myc-tagged hTERT. Cells were incubated with 500 μM H2O2 for 3 h or were preincubated with 10 ng of leptomycin B (LMB) per ml for 30 min followed by 500 μM H2O2 for 3 h. (Upper panels) Nuclear staining with DAPI (blue). (Middle panels) hTERT-myc staining (red). (Lower panels) Tubulin staining (green). Results are shown from five different transfected dishes. Cells with predominantly nuclear hTERT staining were counted by three independent investigators. The quantification is shown on the right side. (B) 293 cells overexpressing full-length, myc-tagged hTERT (hTERT-myc) were preincubated with 10 ng of LMB per ml for 30 min and incubated with H2O2 for 3 h. Immunoblotting of the nuclear and cytosolic fractions was performed with an antibody against myc (upper panel), topoisomerase 1 as a loading control for nuclear fractions (middle panel), and HSP70 for cytosolic fractions (lower panels) (n = 4). The bar graphs below show the densitometric analysis (n = 4). (C) Telomerase enzyme activity was measured in nuclear and cytosolic fractions of cells in the presence or absence of 500 μM H2O2 or 500 μM H2O2 and 10 ng of LMB per ml. A representative telomeric repeat amplification product assay is shown. (D) Telomerase enzyme activity was measured in nuclear and cytosolic fractions of cells in the presence or absence of 500 μM H2O2 or 500 μM H2O2 plus 10 ng of LMB per ml (n = 3).