Research Paper Volume 6, Issue 4 pp 248—263

Figure 3. Methylated TRF2 exhibits an altered nuclear staining associated with induction of replicative senescence in normal human primary fibroblast GM9503 cells. (A) Senescence-associated β-galactosidase assays for GM9503 cells at either p15 or p45. (B) Quantification of percentage of young and senescent GM9503 cells with BrdU incorporation. A total of 300 cells in triplicate were scored for either early passage (p21) or senescent GM9503 (p48) cells. Standard deviations from three independent experiments are indicated. (C) Analysis of indirect immunofluorescence with anti-TRF2-2meR17 antibody in GM9503 cells at either p15 or p45. Cell nuclei were stained with DAPI in blue. (D) Analysis of dual indirect immunofluorescence with anti-TRF2-2meR17 antibody (green) in conjunction with anti-BrdU antibody (red). The early passage (p20) and senescent GM9503 (p48) cells were incubated for six hours in growth media containing 10 μM BrdU prior to being processed for immunofluorescence. Cell nuclei were stained with DAPI in blue. (E) Analysis of indirect immunofluorescence with anti-TRF2-2meR17 in conjunction with 100 ng of TRF2 peptide containing either modified or unmodified arginine 17. Cell nuclei were stained with DAPI in blue. (F) Quantification of percentage of cells with altered nuclear staining of methylated TRF2. At least 900 cells in triplicate were scored in blind for each transformed cell line or each normal primary fibroblast cell line at a given passage as indicated. Both HeLa and WI38VA13 are transformed cell lines. Standard deviations from three independent experiments are indicated.