FIG. 4.

BJ cells maintain their telomeric 3′ overhangs at senescence. (A) Overhang protection assay of DNA from BJ fibroblasts at different PD up to senescence (sen). DNA was treated with or without Exo I before being analyzed in the overhang protection assay. To verify approximately equal amounts of input DNA, 1/30 of each sample was run on a 0.7% agarose gel and visualized with ethidium bromide (input). (B) Weighted mean sizes of the overhangs were quantified and are plotted. Results shown are representative of at least three independent experiments. Error bars represent one standard deviation. Cells were cultured in three different growth series until senescence, and DNA from these cells was used for the overhang protection assay. (C) Senescence-associated β-galactosidase staining of BJ fibroblasts at PD 32 and at senescence. (D) Nondenaturing hybridization analysis of telomeric restriction fragments from BJ fibroblasts at PD 40 and 90 (senescence). Undigested genomic DNA was hybridized to ³²P-labeled C-rich high-specificity probe and then gel fractionated in 0.5× TBE. After the gel was denatured, overhang signals were transferred to a nylon membrane and exposed to a phosphorimager screen (left panel). The denatured gel was neutralized and rehybridized to the Alu repeat probe (right panel). (E) Relative amounts of overhangs were calculated by normalizing signals from the membrane (overhang signals) by the Alu repeat signal (representing total genomic DNA) and plotted. Results shown are representative of three independent experiments. Error bars represent one standard deviation.