Research Paper Volume 6, Issue 4 pp 248—263

Figure 7. Dysfunctional telomeres induce altered nuclear staining of methylated TRF2. (A) Schematic diagram of experimental setup. GM9503 cells (p23) were infected with retrovirus expressing various TRF2 mutant alleles at day -6. After the three-day selection ended on day 0, the cells were cultured for 14 days and then subjected to analysis of immunofluorescence (IF) and cellular senescence. (B) Senescence-associated β-galactosidase assays of GM9503 cells overexpressing the vector alone or various TRF2 mutant alleles as indicated. (C) Indirect immunofluorescence with anti-TRF2-2meR17 antibody in fixed GM9503 cells overexpressing the vector alone or various TRF2 mutant alleles as indicated. Cell nuclei were stained with DAPI in blue. (D) Quantification of percentage of cells with altered nuclear staining of methylated TRF2. A total of 1000 cells in triplicate were scored in blind for each cell line. Standard deviations from three independent experiments are indicated. (E) Western analysis of GM9503 cells overexpressing various TRF2 mutant alleles as indicated. Immunoblotting was carried out with anti-TRF2, anti-TRF2-2meR17 or anti-γ-tubulin antibody.